I have cloned a mycobacterial gene in pETM41 vector,
in E coli EXPRESSION SYSTEM, protein expression was there but it is 44 kDa instead of 86 kDa because of premature termination during translation. All happened due to codon shift from Nco1 restriction site (CCATGG), ATG is methionine and the remaining G cause frame shift and leads to premature termination of protein.
Now I have that plasmid and I want to remove this G by Site Directed Dna Mutagenesis.
I have designed SDM primers
30 neucleotides long (15 NTDs behind and 15 NTDs ahead of G to be removed) Tm 83 degree Celsius, GC content 60%, 3% to 5% DMSO, Mgcl2- 0.5Mm, 77ng templete, 25 ul Reaction.
PCR details-
Initial denaturation- 98°C – 2 min
Denaturation- 98 °C – 30 sec
Annealing- 65°C and 68 °C - 30 sec
Extension- 72°C – 10 min
Final extension-72 °C – 10 min . 25 cycles
Using Pfu Turbo enzyme.
I have repeated experiment several times but not getting any amplification doesn’t show any band in 1% agarose gel, I even proceeded toward Dpn1 digestion but doesn’t any colony after transformation.
Please help me out.
why am not getting any band after pcr ?
how do i troubleshoot this problem this problem?
Why i am not getting any amplification and band in agarose gel after doing Site directed mutagenesis PCR to generate point mutant?
I believe that you may have to optimize the PCR reaction
Especially the annealing temperature, usually the researcher calculate this temperature by reference to the Oligo sequences (4° for G/C 2° For A/T) or determine them using bioinformatics tool as Oligo6 program.
However, your calculate temperature could be higher, you have to decrease your annealing temperatures,
Yet you could apply a gradient annealing temperature (50°C to 65°c in the PCR conditions. Some Thermo-cyclers have an annealing temperature gradient program.
You also have to verify that the Oligos sequences do not inter or intra hybridize together that event decreases annealing with the target gene sequences. Indeed the Oligo6 program can help you for this analyze.
I advise you to verify that your Oligos haven’t been hydrolyzed during their storage. PAGE electrophoresis in 15% acrylamides could inform you about this degradation; you should obtain only one band at 15 NTDs after Ethidium Bromide coloration.
Finally you could also verify the Pfu Taq activity (with a positive control), the Vector and the Buffers (DNase contamination), I advise you to use new solutions and new primers dilution.
Good work
Try using an annealing temperature of around 55°C, mutation pcrs are working fine at that temperature though you might have higher Tm.
Goodluck
Annealing temp depends on salt concentration of enzyme so you should use tm calculator which mast be enzyme specific .
Once I was unable to get any product even after playing with the parameters of the reactions. Then I found this protocol and it worked: http://barricklab.org/twiki/bin/view/Lab/ProtocolsMegaprimerMegawhop
You can test the mutagenesis primers in two alternative standard PCR reactions: with T7 forward primer or T7 revers primer. The result will not look nice on the agarose gel becauce the T7 primers will have most probably lower Tm then you primers. But you can cut the desired product from the gel and use it as large megaprimer in mutagenesis PCR.
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