Fill in/Chew back of DNA overhangs

FILL-IN/CHEW BACK OF DNA OVERHANGS

Last Update: December 2006

PREPARE SOLUTIONS

1. 10X Nick translation buffer:

Mix to 0.5M Tris-HCL pH 7.2, 0.1M MgSO₄, 1mM DTT, and 500 mg/mL BSA (Fraction V) (Store at -20^oC)

2. Deoxynucleotide triphosphates:

Mix to 10mM dATP, 10mM dGTP, 10mM dCTP, and 10 mM dTTP

Note: 25ng of DNA, freshly denatured, will be >50% reacted by 2 units of Klenow at 37^oC for 30 minutes. This means that any excess DNA will remain unmodified. The reactions can be scaled up for greater amounts of DNA, but always make sure enough enzyme is present to modify most of the DNA present (incubation times can also be lengthened)

PROCEDURE - FILL-IN

1. After proper digestion and purification of the DNA fragment, fill in 5' end by preparing the following reaction:

1.1 Mix 10 mL of DNA fragment (<1 mg), 2 mL of dNTPs (10 mM of all four), 2.5 mL of 10X Nick translation buffer, 8.5 mL of dH₂O, and 2 mL of Klenow fragment (2U/ mL)

2. Incubate at 37^oC for 1 hour

3. The DNA is ready for downstream manipulations (ligations can be performed directly on the same sample)

PROCEDURE - CHEW BACK

1. After proper digestion and purification of the DNA fragment, chew back 3' end by preparing the following reaction:

1.1 Mix 10 mL of DNA fragment (<1 mg), 2.5 mL of 10X Nick translation buffer, 10.5 mL of dH₂O, and 2 mL of Klenow fragment (2U/ mL)

2. Incubate at 37^oC for 1 hour

4. The DNA is ready for downstream manipulations (ligations can be performed directly on the same sample)

Note: Klenow fragment activities: 5' to 3' polymerase, 3' to 5' exonuclease (lacks 5' to 3' exonuclease -proof-reading activity of native polymerase)

PROCEDURE - KINASE ADDITION OF g-PHOSPHATE TO 5'-OHs

1. Prepare 5X Forward reaction buffer: Mix to 350mM Tris-HCl pH 7.6, 50mM MgCl₂, 500mM KCl, and 5mM 2-mercaptoethanol (Store at -20^oC)

Note: Kinase is not as active as Ligase (DNA Ligation) or Alkaline Phosphatase (Alkaline Phosphatase removal of PO₄ from DNA fragments), so lower amounts of DNA or larger amounts of enzyme should be used for equivalent results.

2. Add the following in a 1.5 mL Eppendorf tube (in the following order): 5 pmol of DNA (~20 ng), 5 mL of 5X Forward reaction buffer, 10 Units of Kinase, ATP to a final concentration of 1 mM, and dH₂O to 25 mL

3. Mix throughly and centrifuge to bring solution ot the bottom of the tube

4. Incubate at 37^oC for 10 mins

5. Stop reaction by HEAT inactivation at 65^oC for 10 mins (adding EDTA to 5 mM will also achieve the same results, but the EDTA will affect further steps)

PROCEDURE - ALKALINE PHOSPHATASE REMOVAL OF PO₂ FROM DNA FRAGMENTS

1. Prepare (if not commercial) 10X Dephosphorylation buffer: 0.5M Tris-HCl, pH 8.5, and 1mM EDTA (pH to 8.5)

Note: 1 unit of Alkaline Phosphatase (AP) can hydrolyze 50 pmol of 5' terminal phosphorylated DNA fragments (3' recessed, 5' recessed or blunt-ended) when incubated at 37^oC for 1 hour. This implies that 25 pmol of DNA are dephosphorylated (at both ends) in one hour by 1 unit of AP at 37^oC

This protocol takes into account the prior preparation of a DNA fragment, such as one isolated from LM agarose (DNA Fragment Isolation from LM agarose)

2. Phosphatase treat appropriate samples (for example: if digested vector can religate, i.e. digested by a single enzyme) by preparing the following reaction: 20 mL of 10X dephosphorylation buffer, 10 mL of Calf alkaline phosphatase (1U/mL) (contains 50% glycerol, so the volume of AP used should never be more than 10% of the final volume), and ~170 mL of Vector + dH₂O (fragment) (assuming ~0.5 mg)

3. Incubate at 37^oC for 1 hour

4. Add 0.1X Vol of 200mM EDTA/EGTA (in this case, 20 mL) and HEAT inactivate at 65^oC for >10 mins

Note: the EDTA/EGTA should not interfere with ligation reactions if only 0.1X Vol of the AP reaction is used in the ligation reaction (no purification step in between, see DNA Ligation), in which case the EDTA/EGTA would have been dropped down to 1 to 2 mM)

Note: when determining the amount of DNA to use: 1) determine concentration by spectrophotometer or better 2) run aliquots (1, 5, 10 mL) on a gel. Determine the amount that is barely visible (EtBr has a visibility limit of about 100-200 ng on agarose gels) on a THIN gel (thick gels will mask some of the signal, specially at low DNA concentrations). Use two to three times more for the AP reaction. For example: if the aliquot that could be detected in the gel was 5 mL, then use 10 to 15 mL and dilute with dH₂O to the appropiate volume (in the example above, to 170 mL with 160 to 155 mL dH₂O)