blunting with no success! - blunt end ligation (Feb/02/2006 )

Hi
I hope you don't mind me writing to you for some advice, I read your reply to blunt end ligation with T4DNAP...I have been having trouble trying to ligate in a fragment gel prified (1.5 kb) into a vector cut with cla I. I always get more colonies on my control than others and even when analysed they are non recombinant any way. I cut the vector O/N and then PCR purify it before blunting it with T4DNApol and then PCR purify again and dephos with SAP before the final PCR purification again. My insert I gel extract with Qiagen kit, I then phosphorylate with T4Kinase and then PCR purify and finally ligate them for 24hr at 14C. I have just realised may be the T4DNApol is for blunting the 3' overhangs rather than 5' as is mine! is that right?
Any suggestions will be appreciated
Thank you in advance

T4 DNA polymerase is a single polypeptide of 114 kD. The enzyme lacks a 5’ > 3’ exonuclease activity, so it is comparable to the Klenow Fragment, but T4 DNA polymerase has a much more active 3’ > 5’ exonuclease. P. O’Farrell describes the action of T4 DNA polymerase and Tris® Acetate Buffer (TAB), which is a universal restriction enzyme and ligation buffer. (P. O’Farrell 1981 BRL Focus 3 (3) 1-3; P. O’Farrell, 1980. MGG 179: 421; and 1981 BRL Focus 3 (4) 6 indicates that the unit definition in the original paper did not agree with BRL’s unit definition.)
T4 DNA polymerase is the product of the bacteriophage T4 gene 43. T4 DNA polymerase has 3’ exonuclease activity in the absence of dNTPs, and polymerase activity in the presence of dNTPs. The 3’ exonuclease or proofreading activity acts on all 3’ hydroxyl termini. Since the enzyme preferentially binds internal regions of DNA, it is very difficult to saturate the 3’ ends with enzyme. May be inhibited by steric structure of the DNA template, such as when having its polymerase activity activated by Gene 32 protein, at which time the 3’>5’ exonuclease activity is completely inhibited. (Amersham pharmacia catalog 1999, p. 109.)

The exonuclease activity is dependent on the enzyme:DNA ratio. The dependence is roughly linear at enzyme:DNA ratios below 6.25 units/µg of DNA (40 nucleotides/min/3’ end). At higher enzyme:DNA ratios, dependence is non-linear and reaches an apparent rate of 120 bases/min/3’ end at a ratio of 75 units/µg DNA.
The optimum pH is 8.0-9.0, with only 50% activity at pH 7.5 and 9.7. The optimum magnesium concentration is 6 mM. The enzyme also requires SH reagents for maximum activity. It is inhibited when the total ionic strength of the reaction mixture exceeds 100 nM.

In the presence of a single dNTP, the enzyme idles between exonuclease and polymerase activity. The addition of all 4 dNTPs will cause polymerization at up to 15,000 bases/min. In the absence of accessory proteins, secondary structure will impede the enzyme.
When labeling, the enzyme’s idling action removes a terminal 3’ dATP about 10 times faster than a terminal dCTP.

T4 DNAP can be used to fill in 5' overhangs to create blunt ends.
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I assume that when you say "PCR purify," you are referring to a PCR clean up kit? While you have a lot of clean up steps, I have found that the cleaner the DNA is for a blunt end ligation, the more successful you will be.
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Gel extraction of fragments greater than 1 kb should be performed immediately after running the gel. Some lots of agarose permanently trap large fragments if the gel is allowed to sit for an hour.
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I recommend BRL's ligation buffer (which contains PEG) 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. (If you do not use hot water, the PEG will require an overnight dissolving step.)
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What is your insert:vector ratio? I recommend a 2:1 ratio for a 1.5 kb insert into what I assume is approximately a 3 kb vector.
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Good luck.