PCR - Trouble (Jun/12/2008 )
Hi, help needed
I have carried out PCR for a specific gene sequence using a specific primer (comprising two primers), I have standarized everything starting frm temp annealing (using gradient PCR from 40-63 temp- got best result at 62C, showing no non specific bands), Mgcl2 conc, DNA conc (one sample was used), DMSO conc (10% worked well for me), Primer conc. Results were very good using one DNA sample, but when the same reaction was carried out using the same reaction mixture, same program, but using different DNA sample, primer dimer was seen , no PCR amplification of that sample also which showed positive result earlier was seen.
I have run my working DNA samples (as I dilute my concentrated DNA sample accordingly, and use this soln as working stock for PCR reactions) two weeks earlier where they showed band. Cud any one help me.
I have carried out PCR for a specific gene sequence using a specific primer (comprising two primers), I have standarized everything starting frm temp annealing (using gradient PCR from 40-63 temp- got best result at 62C, showing no non specific bands), Mgcl2 conc, DNA conc (one sample was used), DMSO conc (10% worked well for me), Primer conc. Results were very good using one DNA sample, but when the same reaction was carried out using the same reaction mixture, same program, but using different DNA sample, primer dimer was seen , no PCR amplification of that sample also which showed positive result earlier was seen.
I have run my working DNA samples (as I dilute my concentrated DNA sample accordingly, and use this soln as working stock for PCR reactions) two weeks earlier where they showed band. Cud any one help me.
I would always use the first sussesful PCR template as a positive control.
If you are amplifying genomic DNA:
1. Check concentration - about 250-500ng will give good results
2. Check quality - any protein or chemical contamination or degradation if it is long PCR.
3. Hopefully same organism DNA.
4. Isolate new DNA.
If using cDNA:
Same as above +
1. check RNA quality (purity, degradation)
2. RT reaction technique
3. If using different tissue/cell, gene expression may be lower or absent or different isoform expression.
..
1. Check concentration - about 250-500ng will give good results
I have used the same DNA working solution but got a negative results. I have isolated the DNA from different plants. Iwud be repeating the PCR again 2morrow hope so I get the result
did u check the lenght of your template?
If too long, may be it wont amplified. Try
also to check the purity so that there are
not too much protein or RNA left in the sample...
this can be done by phenol-chloroform treatment
(X2 times...may be) and ...ethanol precipitation
and wash. Sometime it is also good to start the
PCR reaction by ''hot start''. It helps the denaturation
of the template