I am new to this forum, however, I read countless of threads and judging from the posts I am pretty much convinced, you can help me
Here's the thing: I am trying to insert a PCR-amplified 0.5 kb fragment into a 4.9 kb vector. Both are cut with the same REs (NotI-HF and SalI-HF, NEB). A sequential digest of the vector confirmed that both enzymes work. The cutting sites for the fragment lie within the primer-sites. After the digest, I load every sample on a gel, let it run and do a gel purification afterwards, eluting the purified DNA in small amounts of water. Then, I start ligating with T4 ligase at 4 °C over night. Here, I tried different vector:fragment ratios, like 1:3, 1:5, 1:10, and 1:15. I also tried to ligate at room temperature for one to three hours and overall, it seemed that 1:3 at 4 °C over night worked best for me. Then, I do the heating transformation in XL-blue bacteria, plate them (10 % and 90 %) on LB-Amp, and usually see only few colonies the next day - let's say on the 90 % plate around 20 to 30, which is quite low, however somehow it seems to work.
After DNA isolation, I start the double digest and surprisingly, I only see the cut plasmid - no fragment. However, the plasmid runs at ~ 5.5 kb and not 4.9 kb which might imply that the fragment is present. When I do a PCR with these samples and the corresponding primers, I can clearly see a band at 0.5 kb (plasmid as a negative control shows no band).
So it seems the fragment is present but the digest does not work properly. My next idea was to look for a RE that only cuts within the fragment but not the vector and yeah, yesterday I found one so I would try this on monday but nevertheless, I would like to know what's wrong with the digest.
Anyone an idea?
Edited by Ulyssus, 10 November 2012 - 02:13 AM.