I clone the E2 gene (444bp) in pACT2 vector during PCR the insert amplification was occur but during digestion the insert release is not occur ?
If you did the colony PCR, it is possible that your PCR amplification is due to your gene fragment contamination during the transformation. If you can specifically PCR amplify the insert from your miniprep DNA, the colony you picked could be a forced ligation of a damaged overhang resulting your restriction enzyme site change. If that is the case, you should see a slight size difference of your plasmid in compare with wild type (It is rare both RE sites are changed), and verify it by sequencing.
Hello,
check your restriction enzymes and buffer
Good luck
Please post an image of the gel and I will be happy to help you out.
Be sure to include a negative control in your PCR. This disparity is likely due to contamination. Use primers for sequences in the plasmid, not the insert and you should produce a small fragment from negative clones and a larger +444 bp product from positive clones. You can then use those same primers for sequencing to verify.
I agree with Jay, use vector primers, or find new restriction sites in the vector that flank your insert and try cutting with those. you will either get 444 + the vector bits, or a small band for the vector bits alone.
PCR is very sensitive and you can amplify unligated excess insert that is still present in the bacterial colony.
Use the vector primers, not your insert-specific primers and sequence
Use different flanking primers for amplifying insert or do sequencing using one of the primer used for PCR to check whether you have any insert.
Digestion may be failed due to many reason.
1. Enzyme has lost activity used new one
2. check incubation temperature of enzyme.
3. DNA prep is not good due to high salt content reprecipitate DNA with ethanol in presence of sodium acetate.
Agreed with Amit. however, I would like to know if you used purified DNA or bacterial culture in your PCR reaction.
Rachel
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