Hi, I am having trouble amplifying YAP via pcr with DNA Pol PfU. I am using PfUUltra high fidelity DNA polymerase. I would like to amplify YAP from gDNA extracted from a cell line that does not have good expression.
The reaction mixture is as follows:
1. dH2O 28.5 ul
2. PfU buffer 10X 5ul
3. dNTP 2.5mM 4ul
4. gDNA 100ng 2ul
5. Primer Fw 100ng 2ul
6. Primer Rev 100ng 2ul
7. DMSO 10% 5ul
8. PfU 2.5U/ul 1.5 ul
The reaction conditions are these for 35 cycles:
95°C x 2 min
95°C x 30 sec
63°C x 30 sec
72°C x 2.5 min
72°C x 10 min
4°C x infinity
I attach the drawing of the primers for cloning YAP through EcoRI and NotI restriction enzymes and the PCR result. As for the latter, in the first column after the marker there is the PCR done on the cDNA, while in the last column there is the PCR done on the gDNA.
when you calculated the annealing temperature of the primers did you leave off the 5' cut site bases. you can only use the specific 18 forward bases for this calculation and similarly you must remove the restriction site and non specific reverse bases as these bases cannt bind the template dna in the first 2 cycles of pcr. 63c seems very hot for an 18mer primer so running at a lower Ta should work
Thanks Paul Rutland for this suggestion.
I repeated the pcr lowering the Ta to 55°C but the result is not encouraging (see attachment). I can't understand the problem.
I really appreciate any guidance.
Ok the primers should anneal hotter than this and your extension times look fine so possibly the problem is secondary structure. Your dmso concentration may be a bit high at 10% so possibly try 6% final concentration of dmso and add betaine to a final concentration of 1Molar and anneal at 60c. If your lab does not have betaine ( very cheap from Sigma) then do not add dmso but use high GC buffer from any other polymerase as this will probably contain both dmso and betaine
I tried to repeat the pcr in a final volume of 50 ul. I reduced the DMSO from 10% to 6% and added 10ul of Platinum GC Enhancer (Thermofisher), formulated for specific amplification and improved yields of GC-rich targets.
In the thermocylator I set these parameters:
95°C x 2 min
95°C x 30 sec
65°C x 30 sec
72°C x 2 min 30 sec
72°C x 10 min
4°C x infinity
the denaturation, annealing and extension cycle was repeated for 35 cycles.
The result improved, but only apparently (see the image).
Although there is a clearer band above 1000bp, it is definitely not the amplicon I am looking for. In fact my amplicon should be higher because it is 1527bp. To confirm this, I purified the PCR product and then performed a Sanger sequencing, both with primers internal to the sequence and with primers that I used for cloning which clearly also have the cutting sequences for the restriction enzymes.
I was unable to sequence the pcr product with any primer, except the fw that I used to clone. I did a blast of the sequence obtained from the sanger, but it is absolutely not yap.
I am losing my mind to amplify yap and I really don't know what else to try.
I desperately ask you for further suggestions.
Tnx,
Barbara
Hi Barbara,
I cannot see what the problem is at present but I would like to see an original .abi sequence file in case it gives some hint as to where the sequence is being generated from if you could attach it please.Shortened amplimers can be generated when the polymerase reads across a loop in the amplimer and I would like to check this. Meanwhile it might be wort trying an amplification with cheap ordinary taq polymerase since your dna and primers seem to work so it would just be a check that your primers can amplify the right thing even though the amplimer quality will not be as good
Hi Paul Rutland ,
I'll send you the .abi sequence files.
In the meantime I'll do some more tests with a cheap ordinary taq polymerase, since the last one I did gave me a similar result to the one I had with PfU.
Below are the primer sequences:
YAPwt_FwPrimer_1 5'-CTTCAACGCCGTCATGAACC-3'
YAPwt_FwPrimer_3 5'-ACCCACAGCTCAGCATCTTC-3'
YAPwt_RevPrimer_2 5'-CATCCTGCTCCAGTGTTGGT-3' Primer_yap_Cloning_Fw 5'-TAAGCAGAATTCATGGATCCCGGGCAGCAG- 3’ Primer_yap_Cloning_Rev 5’-TGCTTAGCGGCCGCCTATAACCATGTAAGAAA-3’
thanks,
Barbara
Thank you Barbara for the sequence files. I had hoped that there was some good sequence that would give a clue as to where the problem lies but there is no sequence readable from any of the files. The signal intensity is around 30 which means that all of the sequencing either failed (signal strength of 300 is needed for big dye 3) or that it failed to load on the sequencer. I would expect to see a huge unincorporated dye peak with failed sequencing which is mostly not there so some possibilities are....not enough dna amplimer
not enough primer, sequenced sample lost on purification
or far too much salt left in the cleaned up sequencing reaction so the sequenced sample did not load onto the sequencer.
In general you need 25 ng of template times the length of the template in kilo bases. for a sequencing reaction
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