Regarding smaI restriction enzyme - Blunt end cloning (Jan/25/2005 )
1) Has anyone used smaI as a RE to cut a vector for blunt end cloning?
Background: Was surfing the web for advise, came upon this website:
Was pretty useful, but in the last sentence, he caution about smaI's activity regarding cloning.
So my situation now is I'm trying to cut a plasmid 2862bp with smaI.
I over digest overnight at room temp. Ran the whole product on a 1% agarose, gel purified using QiaQuick gel extraction kit.
omited the CIAP step as I assume that the vector is fully cut since I extracted the single band plasmid DNA from the gel.
currently doing some polishing of the DNA insert with klenow fragment...
2) So do you guys treat the smaI cut vector with CIAP, t4 DNA polymerase or Klenow fragments to blunt it?
Hope you guys can shed some light....
I always treat digested vector with CIAP or, better (in my hands), Shrimp Alkaline Phospatase...
For successful ligation, the insert DNA then has to be phosphorilated, of course, either by usage of phosphorilated Oligos in PCR or by treatment with Polynuceotide Kinase+ATP.
SmaI is not the best RE out there, but I understand that sometimes you'd have to use it since it's your only choice. I'd definately phosphatase my plasmid in order to prevent self ligation. I recently used Antarctic Phosphatase and it workes also very good. Make sure that the ends of your insert have P's by Kinasing them in presence of ATP!!
Sma I has an isoschizomer which may be a better enzyme. If you still need the blunt end, you can just do a fill-in or chew back the overhang.
I juz started with cloning in pUC using SmaI to restrict digest for blunt ends and when i ran the gel got a linear plasmid but dint do dephosphorylation.....and went on to juz try an ligate my PCR products which was amplified using Taq polymerase.....used the NEB ligase and when the ligase buffer was thawed there were white precipiate which was found to be insoluble possibly the white precipitate shud be the ATP....am I correct...i tried to dissolve it dint work....well will the ligase work with this buffer....tried to liagte but then it dint and got no blue or white colonies......can someone try and help me figure out whats happening.....one more thing is it very important to do dephospho.....but in this protocol online there is a protocol which says nothing of dephosphorylation......tell me iam really stuck...
1) You can simultaneously cut with Sma I and dephosphorylate with SAP. This will prevent the Sma I ends of the vector from religating.
2) The white precipitate in the ligation buffer is probably due to magnesium and DTT. This will usually go into solution if you warm the tube to 37C and then vortex it briefly. You may wish to prepare your own buffer.
5x Blunt End Ligation Buffer (BRL Ligation Buffer): consists of 250 mM Tris-HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000. To prepare 10 mL of buffer, weigh 2.5 g of PEG 8000 in a 15 mL Falcon 2097 tube that has been treated with antistatic spray or wiped with a sheet of fabric softener. Microwave a 100 mL bottle of Type I water for 1 minute on High. Add 4.4 mL of hot water and 2.5 mL of room temperature 1 M Tris-HCl, pH 7.6 to the PEG and immediately mix to dissolve. Cool the mix to room temperature and add 500 µL of 1 M MgCl2, 500 µL of 100 mM ATP and 50 µL of 1 M DTT to a final volume of 10 mL. Aliquot for storage at -20°C. Failure to heat the water will result in PEG that will take approximately 24 hours to dissolve. (BRL Technical Bulletin 5224-1 1992.)
3) If your PCR products were prepared with Taq only, then the ends are probably not blunt, and will typically have a single base overhang on the 3' ends (not always A). You will need to polish the ends with a proofreading DNA polymerase such as Pfu, or use a premixed Taq plus proofreading DNA polymerase.