Tips for optimizing and troubleshooting problems with PCR. Solving "No Product" or "Multiple Bands" are covered. Related videos can be found at patharkar.com.
Greetings Rahul, happy to see you posting again! Your past videos have been a huge help to me. I am currently attempting to amplify a 6kb region using a 5x MasterMix and was wondering if you have any recommendations. I've tried several different combinations of annealing temperature and extension time but nothing seems to do the trick. EDIT: After watching the last few seconds of your video, I realized I should probably try varying my enzyme 😅
If you cannot get a 6 KB product. PrimeStar GXL may do the trick. Really clean, well prepared template can also help. There are certainly many things that can be tried. Good luck!
10:28 - I disagree with the null hypothesis! There is no better DNA polymerase than Primestar GXL by Takara. My friend amplified the entire SARS-CoV-2 genome with it - 30 kb, no trouble shooting. GXL has never let me down and it's less expensive than Q5 (which is sooo finicky)
Well that is what we typically hope to do, disprove the null hypothesis. I agree and stated in the video, PrimeStar GXL is the most robust enzyme I have tried. Best, Rahul
I have used the Sigma Extract-N-Amp solutions. They work reasonably well but not nearly as well as CTAB genomic DNA extractions. Here is the link: www.sigmaaldrich.com/technical-documents/articles/biology/extract-n-amp-plant-kits.html
@@RahulPatharkar Hi Rahul a fellow DIY biologist used a simpler kit from IntactGenomics "FastAmp Plant Direct PCR". Just drop a small punch of leaf the size of 1ml tip in the 2x buffer. Add the 2x Taq mix and initial denaturation of 95 for 8 min. Gave very good results. I think that these buffers contain soluble PVP and bME as mentioned in: sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/lam.12196?fbclid=IwAR1R3NRmXTZs7FhGAt_tsbNEUGx63qmJIXWbnDH9GpJyEylTzYFXer4YCwc
You would simple start by adding multiple primer pairs. Often primer concentrations will have to be optimized so that one product does not dominate over the other one.
Greetings Rahul, happy to see you posting again! Your past videos have been a huge help to me. I am currently attempting to amplify a 6kb region using a 5x MasterMix and was wondering if you have any recommendations. I've tried several different combinations of annealing temperature and extension time but nothing seems to do the trick.
EDIT: After watching the last few seconds of your video, I realized I should probably try varying my enzyme 😅
If you cannot get a 6 KB product. PrimeStar GXL may do the trick. Really clean, well prepared template can also help. There are certainly many things that can be tried. Good luck!
10:28 - I disagree with the null hypothesis! There is no better DNA polymerase than Primestar GXL by Takara. My friend amplified the entire SARS-CoV-2 genome with it - 30 kb, no trouble shooting. GXL has never let me down and it's less expensive than Q5 (which is sooo finicky)
Well that is what we typically hope to do, disprove the null hypothesis. I agree and stated in the video, PrimeStar GXL is the most robust enzyme I have tried. Best, Rahul
Do you have a direct PCR protocol for leaf tissue?
I have used the Sigma Extract-N-Amp solutions. They work reasonably well but not nearly as well as CTAB genomic DNA extractions. Here is the link: www.sigmaaldrich.com/technical-documents/articles/biology/extract-n-amp-plant-kits.html
@@RahulPatharkar Hi Rahul a fellow DIY biologist used a simpler kit from IntactGenomics "FastAmp Plant Direct PCR". Just drop a small punch of leaf the size of 1ml tip in the 2x buffer. Add the 2x Taq mix and initial denaturation of 95 for 8 min. Gave very good results. I think that these buffers contain soluble PVP and bME as mentioned in: sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/lam.12196?fbclid=IwAR1R3NRmXTZs7FhGAt_tsbNEUGx63qmJIXWbnDH9GpJyEylTzYFXer4YCwc
@@tapariayogesh Thanks, good to know. Best
can u tell me how to modify my normal PCR mastermix to conduct the multiplex PCR
You would simple start by adding multiple primer pairs. Often primer concentrations will have to be optimized so that one product does not dominate over the other one.