CHLOROPLAST SEQUENCING

[donnahardy2]

Hi Kiran,

You are making good use of your time while you have to wait to rerun the gels. I am attaching the references, which you requested.

Donna Hardy

postmortem toxicology 1.pdf

(170.02 KiB) Downloaded 294 times

[donnahardy2]

postmortem toxicology 2.pdf

(173.24 KiB) Downloaded 284 times

[donnahardy2]

postmortem toxicology 3.pdf

(186.4 KiB) Downloaded 310 times

[donnahardy2]

postmortem toxicology 4.pdf

(217.83 KiB) Downloaded 285 times

[donnahardy2]

Hi Kiran,

The second paper on analysis of toxic alkaloids has a couple of pages with tables that exceed the upload capacity. Let me think about how I can get this article to you.

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam,
Im sorry about not being able to update you about my recent works.This is because the lab wasnt free due to juniors lab tests.
I had tried to do the gel electrophoresis of the original dna sample & to my relief, the gel showed 1 clear band of dna & also 2 streaks of dna (showing fragmentation)..
This gave me a hope that maybe my other 2 samples of PCR would show results on the gel after making the appropriate changes above (changing gel concentration to 0.7% & dissolving agarose in TAE buffer while preparing the gel)..
So im going to run the gel for the other pcr samples tomorrow..
i'l upload those pictures tomorrow..

The picture of the gel with the original dna sample is crossing the science buddies limit of uploading so i'l reduce the resolution & upload it within 10hrs..

[KIRAN DEEPIKA]

Here is the picture of dna sample run on gel (before PCR)..
I loaded 1st 2 wells with Centella asiatica & 2nd 2 wells with Leucas aspera

Only 1 sample of Leucas showed a band...
But both samples of Cetella showed a streak..

Please comment on this & comfort me..
I am worried about my seminar now..
Thank you like always mam..

[donnahardy2]

Hi Kiran,

I'm glad you included a description of the gels, because I am having trouble seeing the bands. But with your description, I think that the results will be promising for the gels of the PCR samples. Can you send gels in black and white instead of color? That way you could send a higher resolution picture.

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam,
I performed the gel electrophoresis for the other two pcr products & was really happy to find that atleast one of the samples showed a band (it wasnt distinct thou).
In the excitement & hurry to extract the dna from the gel & then send it for sequencing after doing all the formalities.
So i couldnt reply to you all these days.

I am still not able to attach the picture of the gel along with this post..
Also i did'nt find any way of converting the gel picture to X ray format such that the bands are visible..
I am sorry about that..
Wish i was good enough in computers to convert my photos & presentation to post you keeping in mind the size restrictions here..
Sorry for the inconvenience..

In these days i extracted the DNA from the gel by the procedure explained in previous post & i sent it for sequencing yesterday..
They said that they would get back to me in few days & mail me the sequence..
Im keeping my fingers crossed & hope that atleast that comes out fine..

I am done fully with the wet lab part..
I am all gearing up to start Bioinformatics part..
I am getting used to the Bioinformatics tools, online now..
Thank you for everything mam..

[donnahardy2]

Hi Kiran,

Congratulations on finished the lab part of your project! That was a major undertaking. I understand the problem with attaching the gels, so thanks for trying. I am very happy that you appear to have results. Please let us know about the results of the sequencing.


Donna Hardy

[KIRAN DEEPIKA]

Hi Donna & Caroline mam..
I have a very bad news & I am feeling very bad about my state.
I had sent the samples for sequncing 4 days ago.. But today that person called me up & told that my DNA was fragmented..
So its useless fragmenting it..
I am stuck right now & dont know what to do..
I am going to loose a lot of marks because of this..
All my friends have almost completed their project.. They suggested me not to go for this project project as its tough..
Now i see no way out..

Can i do something to save my marks..??
Can i continue my project with the genome sequence of other plants??
I am very depressed.. Infact, i dint have face to post this to you mam..
Feeling very bad that i let you both down even after all the help i have got..
Please help me with this one thing & i wont ask for another easy way out..
I have very less time & my marks decides my future..
I had hopes of being atleast in the top 10 this year & study abroad..
Please help.. I have no where else to go..

Thank you & Sorry for letting you down..

[donnahardy2]

Hi Kiran,

This is disappointing news. The problem was not the plant, but the sample that you prepared. If your marks depend on having good results, then you need to repeat the purification procedure again. You did purify DNA, but it was degraded by the time that you sent it to the sequencing company. I think if you had phenol to the first chloroform step, and then avoid delays in further processing, your DNA prep will be much better. Switching plants will not solve the problem.

In the US, a project that is not completed perfectly would be written up with lots of explanation of what went wrong and what the student might do to avoid problems in the future, and the lack of results would not necessarily affect the final grade. Projects that do not result in expected results are very common. The important points are to understand the science and realize what went wrong. If your marks are dependent on actually obtaining good results, then I can see a definite disadvantage to selecting such a difficult project that is very dependent on technique. Please discuss this with your professor and ask if you can submit the project as it is. I'm sure you learned as much or more as the other students. I don't think you did anything wrong, and you were careful to review each protocol in advance. However, your report of a green color in the initial DNA prep suggested that there was some residual protein in the sample, and there were some delays in proceeding, and this could have affected your results.

Please let me know if there's anything else I can do to help.

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam..
I am really in a confused state..
I am not able to think about any solution..
The only thing which i could do to avoid losing a lot of marks is to get another plant sequence & continue with my project..
During my presentation i will admit infront of my lecturers & externals that i screwd up my PCR sample because of keeping the sample for too long & shear stress..

Please help me by guiding me where i could find Rubisco large sub unit sequence of medicinal plants..
Please let me know any plant (which is sequenced) which is of Centella or Leucas family..
Such that i could get its sequence & continue with the project..
If not of the same family, please tell me a similar plant family such that i could continue..
I really dont want to abruptly stop my project.. mam..

Please do this one favour mam.. It would really mean a lot for me & my future..
Thanks tons mam...

[donnahardy2]

Hi Kiran,

Unfortunately, I don't know of other related plants that have been sequenced. I will see if I can find out.

Are you going to try to repeat your lab work? Or, are you going to get a sequence and continue with the evolution part of the project? Please let me know what you thinking about doing to salvage this project so I can help. Also, please let me know what the deadline is.

In presenting results, it is not usually necessary to admit to making mistakes, and I would not recommend that you do this unless you think your professors are expecting this. You should present your results (fragmented DNA) and explain what happened during the experiment to cause the result. It is possible that your lab environment was not ideal for this type of experiment because there were lots of other possible sources of DNA contamination. You did not have phenol to use with the chloroform for the first isolation step, and if you did not use a microcentrifuge (I can't remember if you had one), it's possible that the sample was not pure enough from the first step. The mineral oil you used may have been a source of contamination.

I hope this will help a little. Please let me know what additional information you need.

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam..
I am not going to repeat the lab work as i hav no PCR kit..
The sequencing company cant sequence my PCR product as its fragmented..
If they do sequence then i have to pay up for something that is of no use to me.. The company guy only suggested me not to get it sequenced..
I want a list of plant names (atlest the link for this) which are of same or similar family as Centella asiatica..
Such that i will search online that particular plant's sequence (with your help of course) & then continue with my project..
I wont admit i was totally wrong.. I will tell them where i could have gone wrong.. Atleast then my externals would appreciate my honesty & give appreciate me..
Please help me mam..

If you have anyother solution, please letme know mam..
I just want to continue with a sequence that i get through internet & consider that to be my sequence & go ahead with my project..
But i wont take credit to the sequence that i will work on..
Hope i dont lose too many marks..
Will be waiting for your reply mam..

Thanks for being there for me, mam..

[donnahardy2]

Hi Kiran,

I’m sorry I did not reply yesterday. For some reason, I could not access the website.

I think your approach is a good one. Centella asiatica is in the carrot family, which is a very large group of plants, so I’m sure that you will be able to find some sequences. The following website includes some of the close relatives of Centella. This source state that there are 20 species of Centella, but I could not find the names of those species.

http://bodd.cf.ac.uk/BotDermFolder/UMBE-3.html

I hope this helps.

Donna Hardy

[KIRAN DEEPIKA]

Thank you Donna mam for helping me with the link..
Please tell me how should i go about to find out the Rubisco genome sequence of any one of the related Centella species..
I am a little confused.. Should i search in NCBI?? Please give me a briefing.. I will search the protocol online myself..
I cant ask my teachers so i am a little worried & dependant on you mam..
Craving for your reply..

Sorry for screwing up my project & troubling you so much mam..

[donnahardy2]

Hi Kiran,

Yes, I’m sorry I did not explain. You will need to get the information from the ncbi website.

http://www.ncbi.nlm.nih.gov/guide/howto ... e-seq-org/

Here is the available information for Centella species:

http://www.ncbi.nlm.nih.gov/gquery/?term=centella

As you will see, no Centella species that have been sequenced, so it too bad that you were not able to do the sequence for your project, as this does need to be done. You can try searching for other related species. It looks like there is some information available on Daucus.

I recommend that you also review the information on the sciencebuddies website for this project again send an e-mail inquiry to Dr. Baysdorfer with your questions.

https://www.sciencebuddies.org/science- ... p012.shtml

I think you should be able get enough information from the published literature to complete your project. You might not be able to find exactly what you want, but there should be enough information for you to present a complete project. Let me know if you can't access the website, or if you need more information.

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam..
I found in NCBI that Centella asiatica rubisco large subunit sequence is already found out..

This is the sequence name & the link-

Centella asiatica voucher PS1212MT04 ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene, partial cds; chloroplast

http://www.ncbi.nlm.nih.gov/nuccore/GQ436636.1

Can i use the same sequence or what mam..

Thank you for this help.. I am not worried about my project any more..

[donnahardy2]

Hi Kiran,

You are better than I am at finding things on this website. Yes, you should definitely go ahead and use this sequence to complete your proejct.

Best regards,


Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam..
Thanks for approving this mam..

I will start with the Bioinformatics part tomorrow..
I cant thank you enough mam..

[KIRAN DEEPIKA]

Hi Donna & Caroline mam..
Sorry for no posts all these days. I had exams & i did well in all my subjects..
My exams got over yesterday..
My project is almost done.. But i dont know how to interpret the results from the cladogram obtained from ClastalW..
Can you please give me a link which would teach me how..

After interpretation of cladogram,We are supposed to present our project the next friday..
Thanks for all the help till now mam..
It meant a lot for me..
Looking forward for your reply..
Hoping to mail you my ppt after next week..

[KIRAN DEEPIKA]

Hi Donna mam,
Can you please letme know which tool is better for obtaining a cladogram.. ClustalW or Phylip??
I will be awaiting your reply..
Please help me..
My final presentation is 6 days from now..

[donnahardy2]

Hi Kiran,

I do apologize for the delay and I missed your first request. I am short of time this morning, but I will reply tomorrow. I will do my best to help you as soon as possible.

Donna Hardy

[donnahardy2]

Hi Kiran,

Congratulations for finishing all of your exams and for doing well! You have worked very hard these last few months and I’m sure this will help ensure a successful future for you.

I am definitely out of my area of expertise in trying to answer your last question, but I have found some information that I hope will help you. ClustalW and Phylip can both be used to obtain a cladogram, and there is no clear consensus on which method is better. For your presentation, you should understand how the programs work.

Here are two websites that provide additional information on these programs:

http://www.cbs.umn.edu/courses/katagiri ... PHYLIP.pdf

http://www.ddbj.nig.ac.jp/search/help/c ... _help.html

I recommend that you follow the procedure on the sciencebuddies guide for this project. This involves using BLAST to find plant species that have the maximum identity to your sequence. You can then follow the step-by-step procedure using ClustalW to construct your cladogram using the sequence of the related plants you have found.

https://www.sciencebuddies.org/science- ... p012.shtml

You can try Phylip if you have time. The information I found on this program indicates the interface is not the best, but it does work and it’s free.

Please let me know if you are able to complete the cladogram, or if you have any problem.

Good luck!

Donna Hardy

[KIRAN DEEPIKA]

Hi Donna mam..
Thanks for the info on this as well..
Im happy to know that you liked my hard work..
My teachers told me to use Phylip instead of ClustalW..
So i had to learn that & use it myself..
I'm preparing my presentation these days..
Hoping to clear all doubts before thursday before i go for my presentation on friday..
I have made a list of doubts.. i will post you in 12 hours..
will ask all doubts at a time..
I will try searching the answers myself but approach you for only the difficult ones..
Thanks for your immediate help always..
I can never forger ScienceBuddies & your Help mam..

[donnahardy2]

Hi Kiran,

If your teachers told you to use Phylip instead of ClustalW, you are very wise to use Phylip. I could not find any information that would suggest an advantage of one program over the other, but perhaps your teachers are aware of a significant difference. I'm very impressed that you were able to learn this on your own so quickly so you can complete your project. I will be ready to reply to any inquiries that you post, as I know you are working on a deadline.

Donna Hardy

[donnahardy2]

Hi Kiran,

I am following up to find out how your presentation went. Please let me know.

I was very impressure with how hard your worked on this project, as well as how thorough you were and how well you planned your experiments in advance. I enjoyed working with you, and I'm sure you will be successful in the future.

Donna Hardy

[KIRAN DEEPIKA]

Hi donna mam.. sorry abt not posting anything all these months.. my presentation went very well.. & i secured 82%.. without your help nothing would have being possible..
Hats off to your constant help, your support when i went wrong & your humility...

Thank you from the bottom of my heart for all that you did to me.. i got placed in a very good company.. There is no internet access.. So could not tell you how much your help meant to me..

Im really impressed with Science buddies to have such people who help students like us..

Thanks again..

Thanks for your blessings as well.. My future seems to be very secure now..
I will never forget your help to me..

Your the best..

[donnahardy2]

Hi Kiran,

Thanks for your message! I am so happy to hear from you again, and to learn about the excellent position with your new company. I'm certain that with your hard work and diligence, you will have a very successful career. I really enjoyed working with you. Good luck to you in the future!

Best regards,


Donna Hardy