Hello everyone, i am trying to amplify a 3kb insert with 64% GC content from a plasmid to be used for downstream cloning purposes. My lab does not do much cloning so we only have SYBR green taq as a DNA polymerase. I did a gradient PCR but the gel showed me that the amplified product is between 1.5 kb and 2 kb.
I double checked my primers and they should not be giving me non-specific products. Can the problem be with the sybrgreen? Would you recommend using an enzyme like Phusion High Fidelity DNA polymerase?
I also read that high GC content of the sequence you want to amplify might lead to problems with PCR, but I could not find what is meant by 'high'. Would 64% be considered high?
Thank you to everyone in advance, I just feel so lost!
What % gel are you running?
What is the voltage set at?
Make sure you're using the correct ladder and marker to ensure they read to 3 kb.
Hello, thank you for your reply! I am running a 1% gel at 90 volts (typically for 45 minutes) with a 1 KB DNA ladder
You're welcome! Whenever I have issues like this, it's usually related to the gel and not the PCR. I wonder what would happen if you increased the running time a little and ran it for 1 - 1.5 hours? I usually run gels for at least one hour to give them time to resolve. I hope this helps!
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