We found your mail on a internet forum where you adviced about Long PCR's, could you help us?
We are trying to amplify a 12.5 Kb fragment using Expand Long Template PCR System from Roche.
We are using a Wizard Genomic DNA purification Kit, Promega or manual extraction with CTAB. Finally we add 500ng of template.
To improve the amplification we added DMSO, other times BSA, but we don`t have any product.
In all amplifications we used the three buffers provided with the Expand Long Template PCR System from Roche, also added MgCl2, until now not product was obtained.
We are thinking that our problem could be:
1- DNA integrity- With which method or kit did you extract DNA for this reaction?
2-Cycling conditions
3- Visualization in agarose gel- You use a neutral or alkaline buffer to run the samples? We are running the samples at 90 V for 1 hr 45 minutes, can you tell us your conditions?
4-Others
The cycle consists in:
93C 2 min 1 cycle
93C 30 sec 35 cycles-45 cycles, we also tried adding time as in your work.
46-68C 45 sec (we have tried with several temperatures in this range)
68C 12 min
68C 10 min 1 cycle
Primers Tm are 68.1 and 69.2, and they are 23 and 27 nucleotides long, respectively, a colleague suggested we should used primers purified by PAGE, is this necessary? We used purified primers after trying some time with the regular ones.
Which do you think are the most critical steps/conditions for this PCR to work? DNA extraction? Conservation? We tried several enzymes: Phusion, and DyNAzyme EXT from Finnzymes, and actually Expand Long Template PCR System from Roche.
We look forward to receiving your advice and suggestions to detect where the problem is.
In advance, thank you very much for your help
Best regards,
Dra Gabriela Sansó, PhD
Centro de Investigaciones Endocrinológicas (CONICET)
Hospital de Niños "R. Gutiérrez"
Gallo 1330, C1425EFD
Ciudad Autónoma de Buenos Aires
Argentina
TE 4963 5931 ext 108
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