CHLOROPLAST SEQUENCING

[donnahardy2]

Hi Kiran,

I will answer the electrophoresis questions a little later today, but I wanted to respond to your question about sending the power point. I am very interested in seeing your presentation.

To upload files, put them on your desktop, then click on the upload attachment tab on the bottom of the post a reply window, then browse your desktop and add the file, then click on upload attachment. The file size is limited, so you may have to do it a few slides at a time.

I will ask if there is a alternate way of doing this.

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam..
There is no UPLOAD ATTACHMENT tab below POST REPLY..
I read da FAQ in ScienceBuddies.. It said i have to obtain permission from board administrators, only per group / per forum can attach images..

Please help.. I really want you to see my presentation more than my teachers..
Looking forward for your help..
Thank you mam..

[carolinethorn]

Hi Kiran,

On my browser I have to click on "Post reply" and then when the white reply window comes up, under that is another blue window with 2 tabs "options" and "upload attachment". Click on the attachment tab and it should be as Donna describes.

Glad to hear things are going so well. I too am interested to read your slides.
-Caroline

[donnahardy2]

Hi Kiran,

Try to relax; you are almost through with this challenging project!

I think you had mentioned previously that you had run a gel, so and I don’t think you will have much difficulty with this part of the project. I would recommend letting the agarose gel set for about an hour to make sure it is firm enough to work with. You should check out the gel box to make sure it will run before you load the gel. Do you have a 100 bp DNA ladder marker to use so you will have a control?

What brand of gel box and power supply will you be working with?

If the electrophoresis works well, you will have a single band in the plant samples and no bands in the negative control. If you have more than one band in the samples and any bands in the negative control, then you will have to decide what to do. Let’s hope for a single band.

Electroelution requires a special device, so if this is not available in your lab, you will need to use the 0.2 % agarose well technique to recover your samples. The procedure that your teacher gave you looks good. If there is an electroeluter available, it would be easier, because you would just need to cut out the section of gel containing the sample, and use the power supply to run the sample out of the gel and collect it in piece of dialysis tubing. Please let me know the brand and model of the elelctroeluter if you have this available and if you have any questions.

Here are the directions for sending the sample for sequencing to the science buddies volunteer who will be doing the sequencing. I had not noticed this before, but you should have e-mailed Dr. Baysdorfer before you started. Also, I had not noticed that he will only accept one sample. Go ahead and send an e-mail to Dr. Baysdorfer and ask him about his schedule and whether or not he can accept two samples. The samples can be sent at ambient temperature, but I think it may take more than 2 days to arrive from India, so you may need to send the samples on ice. You will need to fill out a commercial invoice to export the samples, so let me know if you need help with this part of the project.

1. Before beginning this science project, email Dr. Baysdorfer at California State University, East Bay ([email protected]). Inform him that you are a student doing the Science Buddies Chloroplast Sequencing Project. Give him your best estimate of when you will be sending him your sample and ask him if that is a convenient time for him to sequence it. Remember, he is doing the sequencing on a volunteer basis and you may have to adjust your experimental timeline slightly to accommodate his schedule.
2. Once Dr. Baysdorfer gives you the okay, proceed with the experiment as outlined above and mail him the sample. (The sample is the half of the PCR product you have been keeping in the refrigerator.)
a. Dr. Baysdorfer's reply will contain the address to which you should send the sample.
b. Send the sample via overnight shipping (FedEx, Express Postal Service Mail, or a similar service). The sample will be okay out of the refrigerator for the 24–48 hours it takes to mail it.
c. Clearly label the sample with species name, and which primer mix was used for the amplification.
d. Dr. Baysdorfer can only accept one sample for sequencing.
3. When sequencing is complete, Dr. Baysdorfer will send you the sequence by e-mail.
4. Alternatively, if you have access to sequencing facilities through a mentor or a company that you can pay to sequence your samples, you may choose to do the sequencing yourself.
a. If this is the case, there is no need to contact Dr. Baysdorfer.
b. You may sequence as many samples as you see fit.

Good luck tomorrow!

Donna Hardy

p.s. I sent a message to the sciencebuddies staff to inquire about your power point slides, but I'm sure we will have to wait until tomorrow for a reply.

[KIRAN DEEPIKA]

Hi Donna mam..
I have attached my powerpoint presentation to this post..
Hope you like it..

Thank you Caroline mam for helping me with the attachment problem..
I am hoping you would like it too..
This presentation is made just to show my teachers where i am heading..
I will make the final presentation after finishing my project say by april 2nd week..
Wish to add Caroline mam's name as acknowledgement in the final presentation..

Thank you for all the love to your unseen student..
I will be awaiting both your replies about my presentation..
Criticism accepted..

[KIRAN DEEPIKA]

Wow.. I feel so relaxed already..
Thank you Donna mam for comforting me..
I will answer all your questions by evening..
I wont be doing Gel Electrophoresis TODAY as the labs wont be free..
I will do it tomorrow..
There is no Electroeluter available in our lab.. So I will go ahead with the alternative procedure..
I would want to send 4 to 8 samples for sequencing.. So I dint contact Dr. Baydorfer..
I had read about contacting him before but sequencing 1 sample wont do us good as i am using 2 plants.. Also shipping from India may cost me more which I am not familiar with..
So I contacted a company in India itself & they are gonna charge Rs.550 per sample..
If you feel this is too costly, please suggest me another means.. Or if you say that Dr. Baydorfer can sequence more than 4 samples, then I will mail him..

My presentation is 8MB.. I am not able to attach this file..
So I will be converting it into low resolution PDF files..
Feeling bad that the quality will be reduced & the animation wont be there..
Anyways I am happy that at least I am able to send them to you..
If there is any other means of sending 8+ MB files, please letme know mam..
Thank you for your co-operation..

[KIRAN DEEPIKA]

Hi Donna mam & caroline mam..
I tried my best to reduce the resolution of each of my slides & convert each of them to pdf files..
But few are over 256kBs.. which is crossing the limit of attachments..
Please letme know how i can attach atleast 1MB files..
That way it would be more convenient for me..
Sorry for the trouble..
Thank you for all the help..
Looking forward to mailing my presentation to you..

[KIRAN DEEPIKA]

Hi Donna mam..
I have a problem understanding the order form of a DNA sequencing company..
My project guide is attending a workshop out of town.. So there is no means to mail this to her..
Please help me in filling this form..

Please help me by giving me some pdfs & seminar papers & links related to Forensic Toxicology & Osteoarthritis..
I have to present a seminar also by april 10th..
More than the project, i am worried about the seminar..

If i finish with gel electrophoreses, i will be relieved..
Please have a look at the order form..

Sorry for so many demands..
Thanks a million tons for the help..

[donnahardy2]

Hi Kiran,

I will answer other questions later in the day, but please tell me more about your seminar on forensic toxicology and osteoarthritis. This sounds like two separate topics to me. I have searched the scientific journals I have access to, and cannot find information on both of these topics together. Please explain what you are trying to accomplish in your seminar.

I will work on order form later.

Your presentation attachment didn't make it. We will have to wait for a reply from the science buddies staff for that, hopefully later today.

Donna Hardy

[KIRAN DEEPIKA]

Hi donna mam..
We are supposed to present a 20 mins seminar on each of the topics..
Sorry for confusing you by not being clear.. These two topics are not together..
These carry marks..
So i came to the best place for knowledge & resources & help..

I will wait for your reply on the order form..
Few basic things i know how to fill..

I couldnt send the presentation as each slide was over 256KBs.. So it couldnt attach & also zip files were not allowed..
Will be waiting for your reply on these today..
Sorry for burdening you with my work..
Thanks again...

[donnahardy2]

Hi Kiran,

Thanks for the clarification. I will look for references on the two topics separately.

Donna Hardy

[carolinethorn]

Hi Kiran,

I can walk through the sequencing form with you but don't have time today to talk about your seminar topic.

The top section is your contact information. Date of submission would be when you send it. Completion date i expect they fill in. Make sure you write your email address clearly if you are choosing for them to send the data via email.

Type of service: i would circle "FragmentAnalysis"

Biohazardous : No

The next section is telling you what to send and how to send it.
So you are sending PCR product and primers.
You will need to estimate how concentrated your PCR sample is. You can do this with the spectrophotometer method you used before.

You will need to send the primers are the correct dilution - your saved concentrated stock concentration was 100micromolar ie. 100micromoles per litre. That is the same as 100nanomoles per millilitre and 100picomoles per microlitre. They want you to send it at a concentration of 5 picomoles per microlitre. So you will need to dilute 1:20. You need 10microlitres per sequencing so 20 microlitres total. I think its helpful to send a little extra to account for pipetting errors and loss, so I would dilute 1.5 microlitres of stock primer with 28.5 microlitres of double distilled water. Label it clearly (see my note below about naming tubes). You will also need to do this for the other primer.

Page 2: This table is like a key to know what is in each of the tubes you are sending.
Sample name will be what you have written on the top of each tube. You have 2 samples (one for each plant). Try and label them with a short name that is easy to tell between them but also to identify you (ie. don't just call them sample one and two or they may get mixed up with someone else's). I usually use my initials and then a code for the sample. eg. CFTs1 or KDs1

Your sample type will be PCR product

The concentration you will calculate by testing some in the spectrophotometer (post if you need help on this again)

Template size is the estimated size in base pairs.

Forward primer - write the name you used on the tube.

Reverse primer - write the name you used on the tube.

If you know if there are GC repeats in the region you are sequencing you would indicate that here. I don't think we know that. It can make the sequencing more tricky. But don't worry about that now.

Then if you purified the band from the gel write yes.

Finally there is the space for the photo of the gel. So, remember when you do your electrophoresis to take an extra picture to send with this as well as taking one for your lab notebook. (and maybe a third for using in a presentation)

Hope this helps,

Caroline

Thanks for wanting to acknowledge me. My full title is Caroline F. Thorn, Ph.D., and I am a scientific curator at Stanford University.

[KIRAN DEEPIKA]

Hi Caroline mam..
Thanks tons for literally spoon feeding me with the way you explained the order form..
All my doubts have vanished..
I am honoured to get your time to type a post for me..
If not for you & most importantly Donna mam, i would have gone wrong in a lots of steps during the course of my project..
Wish i had approached ScienceBuddies 4 years ago.. My life would have been dedicated to research then..

I will definately follow all your details about the order form.. & wait for your reply on my seminar topics..
I will incorporate your name now itself for the pre-presentation slides..
I am craving to send my powerpoint presentation to both of you mam..

Thanks again..
Looking forward for your further assistance..

[carolinethorn]

You are welcome for the assistance. And don't forget, you are already very advanced with what you are doing and so very organized and ask all the right questions. I was in my second year in college before I got the kind of challenging project you are doing already and for many they don't get the chance to really do such independent research until grad school!
-Caroline

[KIRAN DEEPIKA]

I spoke to the company people with my teachers assistance to find out in what form they want our sample for DNA sequencing..
The company guy said i have to purify the PCR product & then send the sample for sequencing..
We are supposed to use PCR amplification kit which i was not prepared for..
Also the budget is going over double my estimate..

The PCR amplification kit costs 3250 rupees while sequencing costs 550 rupees per sample.. & 4 samples costs us 2200..
My school will fund me only 50% as it is a part of the curriculum..
Feeling depressed with the cost..

I am sorry for this sad post..

[donnahardy2]

Hi Kiran,

Yes, I agree that Caroline did a very nice job explaining how to fill out the form.

The unexpected expense is not good news. I do not understand why you have to purchase another PCR kit; you have already amplified the DNA and purchased one kit. Is this a requirement of this sequencing company? Or is there a technical reason? Will the company accept your samples if they have not been run on their kit?

I think you should run your gels tomorrow and see what your product looks like. If the gels look OK, then you need explore other options for getting the sequencing done. Send an e-mail message to Dr. Baysdorfer and ask if he can accept one sample and find out how much it would cost to ship a sample. Contact the company that you purchased the PCR kit from, and ask if they have any ideas for sequencing. Contact your local university and find out if they have sequencing equipment and ask if you can use it. There must be a less expensive option. One thing you will need to learn in doing research is to be very adaptable and flexible, because there is always something that seems to go wrong. This is a learning experience for me, because I was not expecting this problem at all.

Also, you should remember that your DNA samples can be stored in the freezer for a while. You can wait to get the sequencing done until you explore your other options.

Donna Hardy

[donnahardy2]

Hi Kiran,

For your upcoming presentation on osteoarthritis, it seems that you should start with information from a basic medical test on osteoarthritis to give you background information. Then, you could add information from the recent literature on specific topics, such as diagnosis, treatment, hip replacements, etc. Here are some recent articles on this topic. This is a very vast topic, so you will have time to just cover the basics. Let me know if you need any of the reference information.


1.
OARSI recommendations for the management of hip and knee osteoarthritis, Part II: OARSI evidence-based, expert consensus guidelines
Osteoarthritis and Cartilage, Volume 16, Issue 2, February 2008, Pages 137-162
Zhang, W.; Moskowitz, R.W.; Nuki, G.; Abramson, S.; Altman, R.D.; Arden, N.; Bierma-Zeinstra, S.; Brandt, K.D.; Croft, P.; Doherty, M.; Dougados, M.; Hochberg, M.; Hunter, D.J.; Kwoh, K.; Lohmander, L.S.; Tugwell, P.
Cited by Scopus (150)

2.
OARSI recommendations for the management of hip and knee osteoarthritis, Part I: Critical appraisal of existing treatment guidelines and systematic review of current research evidence
Osteoarthritis and Cartilage, Volume 15, Issue 9, September 2007, Pages 981-1000
Zhang, W.; Moskowitz, R.W.; Nuki, G.; Abramson, S.; Altman, R.D.; Arden, N.; Bierma-Zeinstra, S.; Brandt, K.D.; Croft, P.; Doherty, M.; Dougados, M.; Hochberg, M.; Hunter, D.J.; Kwoh, K.; Lohmander, L.S.; Tugwell, P.
Cited by Scopus (71)

3.
OA clinical trials: current targets and trials for OA. Choosing molecular targets: what have we learned and where we are headed? • Review article
Osteoarthritis and Cartilage, Volume 17, Issue 11, November 2009, Pages 1393-1401
Hellio Le Graverand-Gastineau, M.P.


4.
Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis
Osteoarthritis and Cartilage
Blagojevic, M.; Jinks, C.; Jeffery, A.; Jordan, K.P.


5.
The effects of oral glucosamine on joint health: is a change in research approach needed? • Review article
Osteoarthritis and Cartilage
Block, J.A.; Oegema, T.R.; Sandy, J.D.; Plaas, A.


6.
Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects
Osteoarthritis and Cartilage, Volume 10, Issue 6, June 2002, Pages 432-463
Hunziker, E.B.
Cited by Scopus (484)

7.
Aging and osteoarthritis: the role of chondrocyte senescence and aging changes in the cartilage matrix • Review article
Osteoarthritis and Cartilage, Volume 17, Issue 8, August 2009, Pages 971-979
Loeser, R.F.
Cited by Scopus (3)

8.
Glucosamine/chondroitin combined with exercise for the treatment of knee osteoarthritis: a preliminary study
Osteoarthritis and Cartilage, Volume 15, Issue 11, November 2007, Pages 1256-1266
Messier, S.P.; Mihalko, S.; Loeser, R.F.; Legault, C.; Jolla, J.; Pfruender, J.; Prosser, B.; Adrian, A.; Williamson, J.D.
Cited by Scopus (10)

9.
Systematic review of non-surgical therapies for osteoarthritis of the hand: an update • Review article
Osteoarthritis and Cartilage, Volume 17, Issue 10, October 2009, Pages 1263-1268
Mahendira, D.; Towheed, T.E.


10.
TGF-beta signaling in chondrocyte terminal differentiation and osteoarthritis • Review article
Osteoarthritis and Cartilage
van der Kraan, P.M.; Blaney Davidson, E.N.; Blom, A.; van den Berg, W.B.


11.
Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage • Review article
Osteoarthritis and Cartilage
Rutgers, M.; van Pelt, M.J.P.; Dhert, W.J.A.; Creemers, L.B.; Saris, D.B.F.


12.
The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-@a and MMP13
Osteoarthritis and Cartilage, Volume 17, Issue 4, April 2009, Pages 464-472
Jones, S.W.; Watkins, G.; Le Good, N.; Roberts, S.; Murphy, C.L.; Brockbank, S.M.V.; Needham, M.R.C.; Read, S.J.; Newham, P.
Cited by Scopus (7)

13.
Biological actions of curcumin on articular chondrocytes • Review article
Osteoarthritis and Cartilage
Henrotin, Y.; Clutterbuck, A.L.; Allaway, D.; Lodwig, E.M.; Harris, P.; Mathy-Hartert, M.; Shakibaei, M.; Mobasheri, A.


14.
Risk stratification for knee osteoarthritis progression: a narrative review • Review article
Osteoarthritis and Cartilage, Volume 17, Issue 11, November 2009, Pages 1402-1407
Hunter, D.J.
Cited by Scopus (2)

15.
Biomechanical, structural, and biochemical indices of degenerative and osteoarthritic deterioration of adult human articular cartilage of the femoral condyle
Osteoarthritis and Cartilage, Volume 17, Issue 11, November 2009, Pages 1469-1476
Temple-Wong, M.M.; Bae, W.C.; Chen, M.Q.; Bugbee, W.D.; Amiel, D.; Coutts, R.D.; Lotz, M.; Sah, R.L.


16.
Osteoarthritis cartilage histopathology: grading and staging
Osteoarthritis and Cartilage, Volume 14, Issue 1, January 2006, Pages 13-29
Pritzker, K.P.H.; Gay, S.; Jimenez, S.A.; Ostergaard, K.; Pelletier, J.P.; Revell, P.A.; Salter, D.; van den Berg, W.B.
Cited by Scopus (81)

17.
Clinical review of chondroitin sulfate in osteoarthritis
Osteoarthritis and Cartilage, Volume 16, Issue 1003, October 2008, Pages S19-S21
Uebelhart, D.


18.
Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration
Osteoarthritis and Cartilage
Rutges, J.; Creemers, L.B.; Dhert, W.; Milz, S.; Sakai, D.; Mochida, J.; Alini, M.; Grad, S.


19.
The''placebo''response in osteoarthritis and its implications for clinical practice • Review article
Osteoarthritis and Cartilage, Volume 17, Issue 10, October 2009, Pages 1255-1262
Doherty, M.; Dieppe, P.


20.
Glucosamine but not ibuprofen alters cartilage turnover in osteoarthritis patients in response to physical training
Osteoarthritis and Cartilage
Petersen, S.G.; Saxne, T.; Heinegard, D.; Hansen, M.; Holm, L.; Koskinen, S.; Stordal, C.; Christensen, H.; Aagaard, P.; Kjaer, M.


21.
Atlas of individual radiographic features in osteoarthritis, revised
Osteoarthritis and Cartilage, Volume 15, January 2007, Pages A1-A56
Altman, R.D.; Gold, G.E.


22.
Depth-wise progression of osteoarthritis in human articular cartilage: investigation of composition, structure and biomechanics
Osteoarthritis and Cartilage
Saarakkala, S.; Julkunen, P.; Kiviranta, P.; Makitalo, J.; Jurvelin, J.S.; Korhonen, R.K.


23.
Current concepts in the pathogenesis of osteoarthritis • Review article
Osteoarthritis and Cartilage, Volume 16, Issue 1003, October 2008, Pages S1-S3
Krasnokutsky, S.; Attur, M.; Palmer, G.; Samuels, J.; Abramson, S.B.


24.
Biomechanical and biochemical characteristics of the mandibular condylar cartilage • Review article
Osteoarthritis and Cartilage, Volume 17, Issue 11, November 2009, Pages 1408-1415
Kuroda, S.; Tanimoto, K.; Izawa, T.; Fujihara, S.; Koolstra, J.H.; Tanaka, E.


25.
Validation of the Knee Injury and Osteoarthritis Outcome Score (KOOS) for the treatment of focal cartilage lesions
Osteoarthritis and Cartilage, Volume 17, Issue 11, November 2009, Pages 1434-1439
Bekkers, J.E.J.; de Windt, Th.S.; Raijmakers, N.J.H.; Dhert, W.J.A.; Saris, D.B.F.
Cited by Scopus (1)

Donna Hardy

[donnahardy2]

Hi Kiran,

For forensic toxicology, there's a Wikipedia website to get you started on the basics.

http://en.wikipedia.org/wiki/Forensic_toxicology
You should also be able to find a basic textbook on this topic to give you the information you need. Here are recent references on the topic. Most of these articles seem like they would be too detailed to include, since you only have 20 minutes for the entire presentation, but let me know if any of these would be useful to you. Lots of analytical chemistry in this topic.

1.
Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene • Review article
Forensic Science International, Volume 188, Issue 1-3, Pages 1-17
Virkler, K.; Lednev, I.K.
Cited by Scopus (4)

2.
Medical negligence in drug associated deaths
Forensic Science International
Madea, B.; Musshoff, F.; Preuss, J.


3.
Fire investigation and ignitable liquid residue analysis-A review: 2001-2007 • Review article
Forensic Science International, Volume 176, Issue 2-3, Pages 93-110
Sandercock, P.M.L.
Cited by Scopus (3)

4.
Mass and NMR spectroscopic characterization of 3,4-methylenedioxypyrovalerone: A designer drug with @a-pyrrolidinophenone structure
Forensic Science International
Westphal, F.; Junge, T.; Rosner, P.; Sonnichsen, F.; Schuster, F.


5.
Analysis of toxic alkaloids in body samples • Review article
Forensic Science International, Volume 185, Issue 1-3, Pages 1-9
Beyer, J.; Drummer, O.H.; Maurer, H.H.


6.
Distribution of methamphetamine and amphetamine in drug abusers'head hair
Forensic Science International
Lee, S.; Han, E.; Park, Y.; Choi, H.; Chung, H.


7.
An assessment of sensing technologies for the detection of clandestine methamphetamine drug laboratories • Review article
Forensic Science International, Volume 189, Issue 1-3, Pages 1-13
Man, G.; Stoeber, B.; Walus, K.


8.
The forensic evaluation of burned skeletal remains: A synthesis • Review article
Forensic Science International, Volume 183, Issue 1-3, Pages 1-5
Ubelaker, D.H.


9.
Medical malpractice as reflected by the forensic evaluation of 4450 autopsies
Forensic Science International
Madea, B.; Preusz, J.


10.
SNPs in forensic genetics: a review on SNP typing methodologies • Review article
Forensic Science International, Volume 154, Issue 2-3, Pages 181-194
Sobrino, B.; Brion, M.; Carracedo, A.
Cited by Scopus (89)

11.
Factors affecting decomposition and Diptera colonization
Forensic Science International, Volume 120, Issue 1-2, Pages 18-27
Campobasso, C.P.; Di Vella, G.; Introna, F.
Cited by Scopus (34)

12.
Postmortem toxicology of drugs of abuse
Forensic Science International, Volume 142, Issue 2-3, Pages 101-113
Drummer, O.H.
Cited by Scopus (37)

13.
Elemental analysis of white cotton fiber evidence using solution ICP-MS and laser ablation ICP-MS (LA-ICP-MS)
Forensic Science International
Gallo, J.M.; Almirall, J.R.


14.
Validation of new methods
Forensic Science International, Volume 165, Issue 2-3, Pages 216-224
Peters, F.T.; Drummer, O.H.; Musshoff, F.
Cited by Scopus (63)

15.
Forensic applications of isotope ratio mass spectrometry-A review
Forensic Science International, Volume 157, Issue 1, Pages 1-22
Benson, S.; Lennard, C.; Maynard, P.; Roux, C.
Cited by Scopus (48)

16.
Forensic anthropology: developments of a classical discipline in the new millennium
Forensic Science International, Volume 165, Issue 2-3, Pages 185-193
Cattaneo, C.
Cited by Scopus (12)

17.
The neuropathology of infant subdural haemorrhage • Review article
Forensic Science International, Volume 187, Issue 1-3, Pages 6-13
Squier, W.; Mack, J.


18.
Repellent effect of some household products on fly attraction to cadavers
Forensic Science International, Volume 189, Issue 1-3, Pages 28-33
Charabidze, D.; Bourel, B.; Hedouin, V.; Gosset, D.


19.
Forensic imaging of projectiles using cone-beam computed tomography
Forensic Science International
von See, C.; Bormann, K.H.; Schumann, P.; Goetz, F.; Gellrich, N.C.; Rucker, M.


20.
Cadaveric volatile organic compounds released by decaying pig carcasses (Sus domesticus L.) in different biotopes
Forensic Science International, Volume 189, Issue 1-3, Pages 46-53
Dekeirsschieter, J.; Verheggen, F.J.; Gohy, M.; Hubrecht, F.; Bourguignon, L.; Lognay, G.; Haubruge, E.


21.
Pharmacokinetics of disappearance of cocaine from hair after discontinuation of drug use
Forensic Science International, Volume 189, Issue 1-3, Pages 24-27
Garcia-Bournissen, F.; Moller, M.; Nesterenko, M.; Karaskov, T.; Koren, G.


22.
Rib fractures identified at post-mortem examination in sudden unexpected deaths in infancy (SUDI)
Forensic Science International, Volume 189, Issue 1-3, Pages 75-81
Weber, M.A.; Risdon, R.A.; Offiah, A.C.; Malone, M.; Sebire, N.J.
Cited by Scopus (1)

23.
Powder method for detecting latent fingerprints: a review
Forensic Science International, Volume 120, Issue 3, Pages 172-176
Sodhi, G.S.; Kaur, J.
Cited by Scopus (24)

24.
Interpreting results of ethanol analysis in postmortem specimens: A review of the literature • Review article
Forensic Science International, Volume 165, Issue 1, Pages 10-29
Kugelberg, F.C.; Jones, A.W.
Cited by Scopus (25)

25.
Quantitative structure-activity relationship (QSAR) methodology in forensic toxicology: Modeling postmortem redistribution of structurally diverse drugs using multivariate statistics
Forensic Science International
Giaginis, C.; Tsantili-Kakoulidou, A.; Theocharis, S.

Donna Hardy






















.

[KIRAN DEEPIKA]

Hi Donna mam & Caroline mam,
I am sorry for my negligence.. I meant PCR PURIFICATION KIT & not PCR AMPLICATION KIT..
The purification kit costs 3250 rupees..
I spoke to my teachers.. They said, this kit consists of eppendroff tubes with seives inside wherein you add sample & buffers provided in the KIT..
This purifies the sample..
This kit can purify 20 samples.. & we need only for 4 samples..
Should i go ahead buying the KIT..?
How long can i keep the DNA in -20degrees..??

The KIT to arrive to school will take 2weeks..
Is it advisable to keep our samples till then??

Or can we not buy the KIT but do the gel cutting procedure which i explained in the previous post..& then send it to him..
I will run the gel today or tomorrow & post you its photo..
Please help. This is really freaking me out..

[carolinethorn]

The DNA is very stable at -20C so don't worry about that. I think it is better to wait to run the gel until you are ready to purify it. But if you have already run the gel it will be ok to keep the slice in the fridge for a few days.

Yes, the purification kits are expensive because of those little sieve tubes. I think you will only need 2 - one for each plant band. Maybe they have product samples they could give you since you don't need a whole kit? Sometimes it helps to ask in writing - including a small description of your project, how young you are, and that you will acknowledge them as a sponsor of your project. If this company says no maybe there is another company you could try to get a free purification kit/few tubes.

There are other purification methods that do not use a kit. I don't remember off hand but could look it up. Is that the bgel procedure you mentioned? Because it would be fine to purify the DNA band by another method and then send for sequencing, i don't think using the company kit would be required.

I am getting a little confused because this thread had got so long and we have all of the seminar topics and the experimental procedures all mixed in together. Sorry if i am already covering something Donna has answered.

-Caroline

[donnahardy2]

Hi Kiran,

Don't buy the kit yet. I will check for alternate methods for you. Caroline has given good advice about waiting to run the gel, but do let us know about the results if you have run the gel.

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna & Caroline mam..
I am sorry for the cause of confusion with seminar & project stuffs in the same thread..
I wont ask anymore doubts concerning the seminar..
Thank you Donna mam for all the information on seminar topics..
I will go through them tomorrow after school & search them through google & ask few which interests me..
Thanks a million for all the hard work for your favorite student.. I am on cloud nine now
My work is totally reduced because searching information is focussed now..

& Caroline mam,
Thanks for your advice on the kit.. I wont buy it like Donna mam said..
So should i perform the gel electrophoresis tomorrow??
& carry out the following steps??

1.Prepare gel.. After solidifying make wells..
2.Run gel to quarter distance with DNA & marker..
3.Remove gel & make a well jst a centimeter below the DNA band & fill it with low concentration agarose gel (0.2%)..
4.Run the gel & allow the DNA to get into the low concentration gel.. & stop the run..
Remove the low concentration gel gently & put it into an eppendroff tube & keep in 60degrees for an hour till it melts..
After melting carry out the isolation part from Phenol:Chloroform step till we get the DNA..
Then send this sample for DNA sequencing..

I found out about the kit.. It will consist of tubes with seives & few alcohol chemicals (which we wont use for anything),like caroline mam said..
But after we finish using the two tubes from the kit, the school would not use the rest of the kit for anything as its of no use for them..
So they wont be funding us..

I will be awaiting your reply on alternative protocols..
I have attached a file on protocols..
Please approve which one of them we can perform tomorrow..
Thanks for all the help..
I am really grateful to both of you..
Thanks millions..

[donnahardy2]

Hi Kiran,

The protocol for your gel electrophoresis step is a good one, and the procedure has lots of detailed information, and should work well for you. Thanks for including the detailed procedure.

DNA samples that are to be used for sequencing must be highly purified. In addition, the samples must be in a specific buffer with a minimum concentration. I recommend that you contact the company that will do the sequencing, and ask what their minimum requirements for purity, concentration, and buffer composition. That would tell you if there is a way to prepare your sample for sequencing without purchasing the kit. I have not been able to find an alternate protocol yet.

Good luck tomorrow!


Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam..
Today we did Gel electrophoresis of PCR product of 1 sample of each plant..
But we did not get any band at all.. Only marker was shown as a streak..
I will upload the picture of the gel i took today..

I realised my mistake after talking to my teachers.. I had used water to dissolve agarose (with ethidium bromide) instead of TAE buffer..
But few teachers told me that wont cause much problems.. But still i am worried a lot..
No bands in the gel made me lose all my hopes..
Felt very bad..
Please let me know what i could do..

Tomorrow i will be running the gel using my isolated DNA & post that picture also..
My teachers feel not optimizing the PCR before using it was the cause of no result.. & keeping the PCR product in -20degrees for 5days denatured the DNA..
Please tell me where i went wrong & what initiative i could take to prevent this..
I have 25uL of 4more PCR products & 15uL of today's 2 PCR products left..

Hope i still have hope to get good results..
All said PCR is very difficult.. Feeling very bad..

To comfort myself i added 3uL of ethidium bromide to 20uL of the isolated DNA(before PCR amplification) in an eppendroff tube to check the presence of DNA..
When i saw it in UV,i found out that it gave a nice orange glow showing the presence of DNA..

Please guide me.. I dont want to let go after so much hard work..

[KIRAN DEEPIKA]

This is another picture..

I will be waiting for your reply..
Sorry i screwed it up.. mam..

[carolinethorn]

Hi Kiran,

Don't get disheartened. Doing all these things the first time is hard. Everyone makes mistakes, the important thing is to learn from them. The first time i poured a gel by myself I forgot to put the combs in it and then wondered where to put the sample!

Don't jump to the conclusion that your PCR didn't work. If you saw nice clear ladder bands on the gel for the markers and no bands then I would say the PCR did not work, but because the ladder marker was not clear I would say it was most likely that the gel was bad. You say you used water in the gel - this is most likely the problem. The buffer in the gel helps conduct the current and ion exchange between the buffer in the tank and in the gel could have caused problems. Do you have any PCR product left to run on another gel with the right buffer in it?

It's hard to see anything on your gel pictures - did you use a filter screen over the UV box? the screen is important for protecting your eyes and face from the UV and also makes the picture come out more like black and white (black gel, white bands) and is easier to interpret. If you did have the screen maybe you need to adjust the camera exposure so that the contrast between gel and bands is better.

Don't get down. You are still doing really well.
best of luck,
Caroline

[KIRAN DEEPIKA]

Thank you caroline mam for comforting me..
I will try to improve the picture quality & then post it to you..
I have a problem in uploading.. I cant upload anthing beyong 256KBs.. I cant upload jpeg files or zip files..
With these constraints i am not able to give you high quality pictures..

I have 25uL of 4more PCR products & 15uL of today's 2 PCR products left..
I had kept the filter screen over UV box.. Teachers suggested me to handle with care..

I will try running the gel (agarose with TAE buffer) again tomorrow with the DNA & the PCR product..
I will take pictures & post it tomorrow..
Hoping we dont go wrong..
Thanks millions..
Still not feeling better.. Hoping to be fine in 24 hours

[donnahardy2]

Hi Kiran,

Your lack of results is disappointing, but please don't worry too much about things you can't control at this point. I am hoping there will be something on your gels tomorrow, both the control and you PCR sample. Running gels will become a little easier everytime you do it. We will be looking forward to hearing from you tomorrow.

Donna Hardy

[carolinethorn]

I'm glad you had the filter for the UV box and took care. Maybe you can get help adjusting the camera a little too so the contrast is better. It doesn't need to be a very high resolution image - i have attached a fairly low res image from another student's project. You can still see the bands clearly even though the ladder bands are a bit out of focus.

Good luck with the next gel!
-Caroline

gelExample.pdf

(212.08 KiB) Downloaded 320 times

[KIRAN DEEPIKA]

Hi Donna & Caroline mam..
Today,I couldnt do gel electrophoresis as there was lab tests for juniors..
Anyways i was going through the seminar papers which you gave mam..

I read the abstracts of all 25 forensic toxicology papers..
I liked-
5. Analysis of toxic alkaloids in body samples • Review article
Forensic Science International, Volume 185, Issue 1-3, Pages 1-9
Beyer, J.; Drummer, O.H.; Maurer, H.H.

12. Postmortem toxicology of drugs of abuse
Forensic Science International, Volume 142, Issue 2-3, Pages 101-113
Drummer, O.H.
Cited by Scopus (37)

Im unable to get the full papers & would like ur help.. If u can,cud u plz send me the full papers??
Please mail the full papers mam..
I would like to study them & do FORENSIC TOXICOLOGY seminar on them..

I stil hav'nt gone through OSTEOARTHRITIS..
I will do it tomorrow & ask you for few full papers on them as well..
Please mail the above 2 as fast as possible as my seminar date is 6th april & its for 50marks..

Thanks you donna mam for everything..