I have been trying to clone a 1600 bp GOI into a pET28a vector. My procedure is as follows: I made 6 reactions for 3 different temperatures and 2 GOIs(same gene different code). 1. add 2 microliters of amplified and PCR cleanup GOI. 2. Add .8 microliters pET28a vector. 3. Add 5 microliters of Q5 Polymerase. 4. Add 1.2 microliters ddH2O. 5. Add 1 microliter of engineered reverse primer. 6. The PCR times at temps are as follows: Initialization step-98C for 2 minutes, denaturation at 98C for 30 secs, Annealing- 55,60,65C gradient for 30 seconds, Extension step at 72C for 7 minutes.
I then ran the products of this reaction on a 1% agarose gel using 2 microliters loading dye and 8 microliters of PCR product. I ran a pet28a plasmid in the farthest right lane. Is it possible the products are too concentrated and thats why the DNA isn't leaving the wells?
If your DNA is not leaving the wells, then you probably are making concatemers during the PCR reaction. Check both the vector and GOI with BLAST to see if there are any regions of partial identity near the ends. Also, consider increasing your annealing temp to just 60 or 65°C instead of a gradient that starts so low.
There are identical sequences of 11 BP starting at 1845 BP of pet28a and 181 of GOI. Another identical sequence at 1219BP of pet28a and 360BP of GOI. There is a very similar sequence(15/16BP) at 1275BP of GOI and 4499 of pet28a. None of these stand out as being exceptionally close to the ends to cause concatemers.
They don't have to be right at the ends, they just have to be stable enough that the overlaps can be extended. After just 2 cycles, you will have 100% identity, and the overlaps can start producing concatemers efficiently. You should probably start by increasing your annealing temp. PCR with long fragments can be a lot of trial and error.
Hi Reece,
You described that your PCR reaction contains your PCR cleanup GOI and pET vector. Why are you doing PCR again with PCR cleanup GOI? and also why are you including the pET vector in your PCR reaction? Also your PCR reaction does not contain forward primer?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques