A tried and true cloning protocol is FAILING miserably! - Molecular biology (Jun/24/2005 )
I have used the following subcloning protocol time and time again with great results...except for now, and I have not idea what is going on!! Here is the basic bare bones protocol:
Cut vector (5.6kbp) with BamHI and SnaBI-->isolate 5.50 kbp fragment, no CIAP treatment
Generate insert by cutting another construct with BglII and Hpa I-->isolate 780 bp band
Ligate (BamHI/BglII compatable choesive; SnaBI/HpaI), using vector/insert=1/3, V=2.2I, 46 ng vector, 21 ng insert.
Transform into XL2 blue cells
LB/Amp100 plates look good (control=5, V+I=12), but restriction digest analysis shows my clones are just vector without insert, or a vecotr the size abut 3.5kbp! I don't know that this species is, and knowing that I think will be the key to what is going on here.
Aside from the insert, I am using all the same regeants(enzymes,ligase, kit to purify DNA) and prep techniques for this construct as for the previous constructs I used this protocol . Any ideas would be great! Thanks.
Is it possible that your agar plate selection agent is bad?
Doubtful (I used the same batch of LB/amp100 plates to check some competent cells, and they worked ok), but at this point I will check into everything that sounds reasonable! We did purchase a new brand of agar to make plates, I wonder if something is going on there....
Thanks for the suggestion...keep them coming!
BamH I can aggregate and become inactive if mishandled or left on ice too long. Perform a test restriction on your vector and make sure that the BamH I is working.
It sounds as if you have a 5.6 kB vector, and are cutting only 0.1 kB from it, and gel purifying. I find it hard to believe you could detect the difference between a single and double cut vector with this length difference. Perhaps (as suggested above) one of your enzymes is not cutting. Cut your vector with the two enzymes separately to make sure you are cutting at both sites. If the sites are too close, there might be a problem in cutting the end left over after the first cut.
You might consider sequencing the weird product to determine what is happening.
did you use EtBr?
I come a little late, I hope I can still help
How long do you digest with SnaBI? I have recently been using it to clone, also with protocols that used to work, and have had big problems, even though everything looked fine on gel.
I solved it by digesting less than one hour with SnaBI. I can't explain why, but it seems that longer digests tend to cause problems.