we cloned a gene in pet28a and got a recominant colony. The insert was 720 bp.
we' ve done colony PCR on colony with forward primer of gene and reverse primer of pet. I got a product about 500 bp on agarose gel. Also, we 've done
nzyme digestion on the recombinant plasmid. The digested plasmid was on incorrect size on the gel. This phenomen can be happen or No?
hello Mahboubi,
can you tell us where the product primer is in the product sequence...is it at the very start so you are expecting a pcr size greater than 720 or is it further into the sequence so a shorter amplimer is expected. What size pcr product are you getting. Also is there an enzyme cut site within your product that was cloned and what were the sizes of the fragments of the cut recombinant plasmid please.
Hello Mahboubi
Can you give details which site you cloned and what restriction enzymes were used. better use gene amplifying primers only for colony PCR.
Yes, the double digested insert had 720 bp and we ligated it in pET28a in position of XhoI and NCoI. Then we transformed it E. coli DH5a. The colony was separated on kanamycin Agar. The Colony PCR were done on it. It is clear, we dont have the end of insert gene. But the forward primer of insert and Terminator T7 of plasmid had a product. We also had the restriction place, because double digestion on recombinant plasmid had two product.
Hello Mahboubi
can you give clear details which one you forward primer and which is reverse primer.
you need to get positive results in colony PCR which carried by using PCR primers dont use vector primer
forward primer of insert and Terminator T7 of plasmid had a product size if its near 200 bp you dont have insert in recombinant vector
if send gel pics it will help full to find problem easily
forward primer of insert and t7 terminator. the product of clony pcr is about 500_750 bp; while we have a product about 780 bp
The best way to confirm would be to send your clones plasmid for Sanger sequencing with the primers you used of cloning. Don't waste your time in confirming the clones as you already did pre-screening.
- Badges
- Science method