Is there anything can be done if the insert gene contains an additional restriction site which recognized by restriction enzyme not only one restriction site at the end of gene sequence, but another at middle of the sequence. In the middle of the experiment when checking for digestion via gel electrophoresis I realize this is the case happened since I find extra band in the gel. (Insert is cut into two parts).
How can I solve this issue if I only have my designed primers for this sequence, one type of polymerase enzyme? (I dont have any other materials which are not needed for this experiment).
Dear Berk Efe Altın
you can:
1) Order a sintetic gene codon optimized to remove the restriction sites and re-do the cloning.
2) Perform a mutagenesis and replace the codons corresponding to the restrction site in the middle of the sequence with different codons codify for the same aminoacids.
3) Use a different cloning approach as the PIPE cloning, which do not require the use of restriction enzimes.
best
Manuele
If you have a lot of template you could try doing a partial digest and then isolating the band of the fragment which has not yet been cut at the internal site. I would suggest doing a sequential digest with the single cutting enzyme first and a reduced incubation time for the second.
if the two parts are fundamental for your gene , please purify the bands from the gel , use the ligase and add adaptors for a new cloning by PCR, then you can re-clone using new enzymes fit for the adaptors
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