Real-Time PCR using genomic DNA (without DNA purification) - (Apr/29/2010 )
Hello. I need to normalize my samples after extraction of the genomic DNA. I will lysate my samples (frozen cell pellet; very low number of cells) following this protocol:
• Add to the cell pellets:
- 100 µl Kawasaki buffer (20 mM Tris HCl, 1.5 mM MgCl2 , 25 mM KCl, 0.5% Tween-20 (v/v))
- 6 µl proteinase K (20 mg ml-1, dissolved in aqua bidest)
• Incubate the samples at 56 °C overnight (cell lysis).
• heat the samples to 95°C for 15 min to inactivate proteinase K.
• centrifuge at 15300g and 4° C for 5 min
• Use the lysed samples directly in the PCR analysis without any further manipulations.
(Harald Lahm et al.. Transgenic Research 7, 131-134 (1998))
Do you know wich kit for the analysis of GAPDH expression can I use for the normalization of my samples?
I found some kits from QIAGEN (SeqTarget LongRange PCR), but I am sure that someone knows a better solution for me.
You might try Finnzymes's Phire polymerase - http://www.finnzymes.com/directpcr/index.html - which I think is included in Dynamo FLASH sybr or probe master mixes - http://www.finnzymes.com/realtime_qpcr/dynamo_qpcr_kits.html. It shoud work without DNA extraction.
vladooo on Apr 29 2010, 11:05 AM said:
Has anyone on this forum used the Phire Animal Tissue Direct PCR kit on mouse tissue (using the direct protocol)? I have been trying to troubleshoot this kit and it has been very unreliable. I've optimized the annealing temperature of my primers (which have been used plenty of times for PCR on purified gDNA), have used more and less tissue, and have tried using the dilution protocol. Is there anyone out there who has had uniform success with this kit on mouse tissue?