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Hi, I have chosen a Science Buddies project for my science fair. Below are a few questions I had while preparing to conduct my experiments for the following project: Investigate Native Plant Evolution with Chloroplast Sequencing. Really appreciate if the experts in this forum can help answer my questions.
My Questions :
1. In the materials list, it says that we need a master primer mix and four sets of primers. What is the difference between a master primer mix and the other primer mixes?
2. In order to get accurate results, do the primer mixes have to be the same brand as the reagents and the Taq buffer or can they vary?
3. What is a negative control for a primer mix?
4. Is a 100 bP DNA ladder required to do this project or would the thermocycler and the software programs on the computer be enough?
5. Which website would be the fastest and most reliable to purchase the consumables for this project?
Question about my biology project, Native Plants Genome Sequencing
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Re: Question about my biology project, Native Plants Genome Sequencing
Hello,
This is a really interesting experiment! To answer your questions:
1. PCR Master Mix is a premixed solution, consisting of Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers, which ensure efficient amplification of DNA templates by PCR. On the other hand, the primer mixes are just composed of a forward primer and a reverse primer.
2. It is fine to use different brands to get accurate results, as the experiment states that any brand PCR reagents should work, as long as the final concentrations are consistent. (dNTP- 200 µM each, MgCl2 (5 mM), Taq buffer (1x), Taq polymerase; units needed for reaction will depend on the exact brand of polymerase; read and follow individual product recommendations)
3. Typically, negative primers are just all the PCR components + water (no DNA).
4. You need to use both the thermocycler and 100 bp DNA. The thermocycler is the apparatus that is used to amplify the DNA in the PCR reaction whereas the 100 bp DNA ladder is used during electrophoresis to make the DNA appear as bands.
5. I would recommend using Carolina Biological Supply Company or Amazon, which are Science Buddies approved suppliers. If you use Carolina Biological, certain materials in this lab will require a teacher to order the chemical and ship it to a school address.
Hope this helps!
C.M.
This is a really interesting experiment! To answer your questions:
1. PCR Master Mix is a premixed solution, consisting of Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers, which ensure efficient amplification of DNA templates by PCR. On the other hand, the primer mixes are just composed of a forward primer and a reverse primer.
2. It is fine to use different brands to get accurate results, as the experiment states that any brand PCR reagents should work, as long as the final concentrations are consistent. (dNTP- 200 µM each, MgCl2 (5 mM), Taq buffer (1x), Taq polymerase; units needed for reaction will depend on the exact brand of polymerase; read and follow individual product recommendations)
3. Typically, negative primers are just all the PCR components + water (no DNA).
4. You need to use both the thermocycler and 100 bp DNA. The thermocycler is the apparatus that is used to amplify the DNA in the PCR reaction whereas the 100 bp DNA ladder is used during electrophoresis to make the DNA appear as bands.
5. I would recommend using Carolina Biological Supply Company or Amazon, which are Science Buddies approved suppliers. If you use Carolina Biological, certain materials in this lab will require a teacher to order the chemical and ship it to a school address.
Hope this helps!
C.M.