I have no priming on the plasmid sequence after Maxi and Miniprep. Even the universal primers do not work. I repeated the plasmid isolation many times with different kits and reagents. No results =(
What is the OD260 and ideally the od260/230 ratio?
Have you run a sample on an agarose gel to see how much dna you have?
Does your plasmid have an insert and what size is it?
How much plasmid DNA are you using in your PCR? It's possible to have way too much (or not enough).
Sometimes, you need to set up a digest with a known single cutting enzyme (that does NOT make a cut in your insert!) to get efficient amplification in PCR.
I recommend digestion mapping using the known RE of your sequence to make sure of the plasmid and the insertion size. Use the uncut plasmid as control
If it worked, you can say it's the pcr that is not working (may be due to not suitable annealing temperature or deficient primers, etc)
Otherwise the problem can be more fundamental, such as starting the cloning with wrong template, unsuccessful cloning, etc.
Check your cloning steps carefully, sometimes small mistakes can ruin the whole process. For instance, if you are using a standard cloning make sure you have dephosphorylated your vector. Happy to help further :)
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