PCR result: Nothing! - Urgent! (Apr/27/2006 )
Hi ladies and gentlemen:
Last week, I select a pair primers Via Primer3.0(Explored by White Head Institute), then add XhoI site on the 5' ends both of the Reverse and forwards primers, besides 4 nucleotides as the anchor nucleotides are added on the 5'end of the XhoI. When I do PCR with this pair of Primers, I get nothing! My Amplicon is 4.3kb. Before that, I Managed to get an ampicon with primers without restriction site overhangs, when I use this PCR product as template and primers with XhoI But without anchor nucleotides, I could get weak bands. now I USE the Primers with XhoI no matter with or without Anchor nucleotides, and Genomic DNA as template, I can not get anything even unspecfic bands.
Human Genomic DNA as the Template.
PCR condition as below(all is final conc.):
Mg2+: 1.5M
dNTP :200uM
Template:500ng
DMSO5%(GC content in AMplicon is 57%)
1X buffer
primers:500nM each
DNA polymerase is DyNAzyme EXT DNA polymerase from Finnzymes
RXNs Program:
94 degree celius 3min for initial denaturation
then 35cycles:
94 degree celius for 25sec
55 degree celius for 30sec
72 degree celius for 4min
then extensive enlongation at 72 degree celius for 10min
Can Anyone told me Why?
Any suggestion is appreciated!
Thanks in advance!
Last week, I select a pair primers Via Primer3.0(Explored by White Head Institute), then add XhoI site on the 5' ends both of the Reverse and forwards primers, besides 4 nucleotides as the anchor nucleotides are added on the 5'end of the XhoI. When I do PCR with this pair of Primers, I get nothing! My Amplicon is 4.3kb. Before that, I Managed to get an ampicon with primers without restriction site overhangs, when I use this PCR product as template and primers with XhoI But without anchor nucleotides, I could get weak bands. now I USE the Primers with XhoI no matter with or without Anchor nucleotides, and Genomic DNA as template, I can not get anything even unspecfic bands.
A quick question: how different are the Tm's of your primers (but only include the sequences that will bind the template, not the XhoI bases)? If they're more than ~6C, you are in for a hard time trying to get a decent product.
If the Tm's are OK, I would try the PCR reactions mixing your old and new primers. If one reaction works, and the other one doesn't, you know it's because of the primer with the RE overhang.
If your PCR is not giving you good results, try changing your annealing temp and do a touchdown PCR.
Why not just use the old primers, which give you a product, and blunt-end ligate? Yes, you will have to screen your colonies, but you should sequence your clones anyway.
Hi ladies and gentlemen:
Last week, I select a pair primers Via Primer3.0(Explored by White Head Institute), then add XhoI site on the 5' ends both of the Reverse and forwards primers, besides 4 nucleotides as the anchor nucleotides are added on the 5'end of the XhoI. When I do PCR with this pair of Primers, I get nothing! My Amplicon is 4.3kb. Before that, I Managed to get an ampicon with primers without restriction site overhangs, when I use this PCR product as template and primers with XhoI But without anchor nucleotides, I could get weak bands. now I USE the Primers with XhoI no matter with or without Anchor nucleotides, and Genomic DNA as template, I can not get anything even unspecfic bands.
A quick question: how different are the Tm's of your primers (but only include the sequences that will bind the template, not the XhoI bases)? If they're more than ~6C, you are in for a hard time trying to get a decent product.
If the Tm's are OK, I would try the PCR reactions mixing your old and new primers. If one reaction works, and the other one doesn't, you know it's because of the primer with the RE overhang.
If your PCR is not giving you good results, try changing your annealing temp and do a touchdown PCR.
Why not just use the old primers, which give you a product, and blunt-end ligate? Yes, you will have to screen your colonies, but you should sequence your clones anyway.
Hi swanny:
Thanks for your kind reply!
The primers is selected by Primer3.0, the Tm difference between the forward primer and revese primer is lower than 0.5 C,
A strange result happens, When I titrate Mg2+ from 1.5 to 1.75mM, THE Specific bands appears!
At the same time, I improve the Annealing Temp from 55C to 60C.
the Tube contains 1.75 mM Mg2+ produce more specific bands than the tube contains 1.5mM Mg2+.
BTW, I was confused that, with The Mg2+ concentration 1.5mM, When I Place The tube on the 52C, 55C and 60C in gradient PCR, only the tube on the 60C column produce the Target band. the other RXNS compnents are identical!
could any one intepret this phenomenon?
thanks!