Hello, I am trying to amplify a 2.3kbp region of murine Fgf9 promoter, using several high fidelity polymerases and several thermocycler runs conditions.
The oligo I used were published, but I needed to change the 5' tail to introduce my restriction sites for cloning.
As first, I tried to use DNA taken by tail biopsies, normally used for genotyping mice... With such DNA, I did several run with Q5U hot start polymerase as well as KOD polymerase...
Both Polymerases did not work, despite I added GC enhancer buffer (for Q5 pol) or DMSO (final conc used from 2 to 5%) for KOD... I tried to use several Mg2+ concentration. I tried, also to do a touchdown PCR moving annealing temperature from 65 to 60 °C (10 cicles) for an higher stringency and then other 25 cycles with 57 °C as Ta... This time a very low rate band of expected size was seen with KOD, but together with many aspecific products...
For Q5U I also used the suggested Ta of 72°C despite with lower temperature nothing was appearing...
Therefore I moved to a new ultrapure DNA source, extracted with a kit from cultured cells (Pan02) and repeated same conditions... again nothing.
Again several conditions tested.
Finally I decided to change also the oligonucleotides and with Q5U in touchdown conditions no bands was appearing.... with KOD instead a broad aspecific pattern was shown on gel... Now I am repeating a PCR with DMSO 2% and high temperature of annealing, without touchdown...
But honestly this is my first time experiencing so many problems with a PCR.
Any advice?
I understand that the primer sequences are published but spelling errors do occur in published work. I would BLAST the primers and check them against the mouse genome and also check the snp database to make sure that there are no snps reported close to the 3' end of either primer . You could even make 2 internal primers giving about 200bp product with your F and R primers to prove that the primers can work on your dna. What is the od260/280 ratio of your dna and how much are you using in your pcr. Are you getting a blurred band at about 80bp indicating a primer dimer
Paul Rutland thank you for your replying.
I also did PCRs with internal and external couples of primers without success, but suddenly ONE of this couple decided to work... And I had success to obtain my promoter. I will do the sequence of the construct once I finished cloning procedure to understand whether SNPs were present preventint other couples to work.
Well done Pasquale Pensieri I am happy that it worked and would be interested in hearing how the sequencing went and if there are any snps in the primer region
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