Hi
I am trying to do a PCR for a 1kb fragment using a plasmid of 11kb as a template. But unfortunately on the gel, the band is as large as the plasmid, that is 11kb.
Would anyone know that that might be coz I used too much of plasmid? Or has seen something similar and has any suggestions.
Thanks in advance
Anusha
have you linearised the plasmid before amplification?
What pcr cycle are you using to get an 11kb amplimer. If you are amplifying an insert from the vector is it certain that the insert is present in the vector. How much vector are you using> 11kb is very small in dna genomic terms so you may just be seeing your vector dna that you added as template and the pcr has failed completely....this seems more likely than your having amplified 11kb of template. Run an un amplified sample diluted as if it was in the pcr mix as a control
I believe that what you see in the gel is actually your template.
If so, then your template is too concentrated and actually inhibiting your PCR reaction.
Try diluting the template 1:100 or even 1:1,000 and try again.
To prevent amplifying the whole vector template - limit your elongation step. There's no way that in 30-60 seconds your polymerase will amplify 11Kb (consult with the manual for your polymerase for adjusting the time of this step).
Good luck!
I would suggest using another set of primers. You might have non-specific binding occurring. Yulia had a good idea as well; often times the template DNA is too concentrated and it can interfere with PCR results. Perhaps the TRIZOL method of DNA extraction might be a better approach, rather than column isolation. Just an idea. Best of luck.
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