BLUNT END LIGATION - DIFFICULTIES ON BLUNT END LIGATIONS (Nov/26/2007 )
HI. I am encountering serious problems with my cloning.
The insert: I digests my plasmid DNA(VP7) with DNASE1, the fragmentation works well I get fragments in the range 50-400bp. I then use T4 DNA polymerase to repair blunt ends and then dephosphorylate with antarctic alkaline phosphate.
The Vector:I use pVII phagemid vector. I cut the vector with PmeI, the vector is cut well.
The ligation: I then add vector:insert at a ratio of 1:3 to the ligation rxn. 1U T4 DNA ligase for 16 hrs at 25 degrees.
I electroporate into competent TG1 cells (Stratagene). But I get only 20 colonies on plates the next morning!
In general i use method described by Gupta and Chaudhary.
Your suggestions will be highly appreciated!
Thank you
Blunt ends cloning always got a lot of troubles. I believe if you read through this subforum (Molecular cloning), you will see many have complained about it and veterans like perseneblue have always adviced against it. So try any way that you can to NOT use blunt ends.
Further, I am curious about your post. You dephosphorylated the INSERT, but NOT the VECTOR???? Since this is blunt ends cloning, I would think that this could cause serious problem, isn't it?
Further, I am curious about your post. You dephosphorylated the INSERT, but NOT the VECTOR???? Since this is blunt ends cloning, I would think that this could cause serious problem, isn't it?
Well I DO NOT dephosphorylate the vector because I include the restriction enzyme PMEI used to digest the vector in the ligation reaction. The inserts I desphosphorylate because since they are blunt ended-they are prone to self-ligation. Your ligation scheme is similar to Stratagene's PCRScript cut with Srf I. Download a copy of their kit's instruction manual. They recommend an insert:vector ratio of 40-100:1 for this sort of vector-recutting method. While it is much higher than the conventional 3:! ratio, it works perfectly. The insert is polished with Pfu and is not dephosphorylated. The ligation reaction contains T4 DNA ligase and Srf I to recut any vectors that religate without an insert. If the ratio of insert:vector is too low, the usual low level contaminating nucleases can nibble the ends of your DNA fragments. Your Pme I may need to be titrated in order to avoid this artifact.
What about your controls? Vector cut with Pme I plus and minus ligase (no additional Pme I in the ligation)? This will telll you if the Pme I is nibbling and they are important controls.
T4 DNA ligase: NEB units or Weiss units? 1 Weiss unit = 67 NEB units for blunt-end ligations.
Ligation buffers with PEG work better for blunt-end ligations. 5x Blunt End Ligation Buffer (BRL Technical Bulletin 5224-1 1992): 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.
A final note: I have been performing hundreds of ligations per year since 1981 with a 99% success rate. Blunt end ligations generate 10-fold fewer colonies than sticky-end ligations when performed carefully. This should not be a problem. Exonuclease contamination of restriction enzymes, BAP and CIP will nibble the ends of your DNA.