Hello everybody, I´m a bachelor´s student I nee dto amplify a 5 kb fragment from a plasmid. Therefore, I perform PCR, and then gel electrophoresis. Subsequently, I extract the 5 kb band and try to re-amplify the 5 kB fragment by doing the PCR under the same parameters. However, the reamplification does not work (I tried quite a few times, even with new primers). The primers which I use should be okay because the first amplification of the fragment worked quite well. There where some unspecific products but I removed them by doing a gel extraction of the amplified 5kB product after gel electrophoresis. The DNA concentration was around 20 ng/µl (with bad correction values). I use TaKaRa ExTaqTM Polymerase for amplification and 1 µl of the above mentioned template DNA.
I would be glad for any help, thanks in advance
Hi,
If you know already that the primers work, why don't you try to change the PCR conditions? In my experience, 2 rounds of PCR do not work very well. I usually got several bands and then by isolating from the gel a part of the yield was lost. I always used RedTaq polymerase for the fine work but I am sure that there are other enzymes you can borrow in your lab (I have no experience with your enzyme but based on the description it should work fine) . See if you can make the elongation time longer and add a few more cycles to increase the efficiency of the reaction. If that doesn't work try changing the enzyme.
Good luck!
Why are you bothering with the reamplification step? Simply amplify the product from the plasmid, do large volume reactions and use a better gel extraction kit.
Do you just get a smear when you re amplify. 10 cycles is 1000 times as much amplimer as you started with. Sometimes over amplification leads to template annealing with template and just getting longer. Try diluting your amplimer 100 times. set up 8 tubes of 1ul of 1/100 amplimer and amplify under previous conditions. after 8 cycles remove 1 tube every 2 cycles and I think that a couple of tubes will have good clean amplimer and the tubes which have the most cycles will look like a long ,high molecular weight smear
Aline, seems like you are using too much DNA as template, hence the low yield and extra bands. Try diluting the plasmid 1:1000 (1 part of plasmid and 1000 of ultra-pure WATER) and using 1 microliter of the dilution as template. Good luck!! You may also want to check this paper out for extra tips on tuning long-distance PCRs: https://www.ncbi.nlm.nih.gov/pubmed/16497393
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology