Preparing Vector for Blunt-end Ligation

(cf: 96:F28)

1) Cut 2-10 mg of Vector with blunt restriction enzyme (e.g. Eco RV)

*In order to insure complete digestion, I use 2-3 units/mg for 2-4 hr, then spike with another 1-2 units/mg for 1-2 hr.

2) Heat inactivate @ 75° C for 10-15 min.

*NOTE* If you are planning to phosphatase your vector, now is the best time.

3) Phenol/CHCl₃ Extract the DNA

Back extract with 1x TE, pH 7.5

Desalt (EtOH pptn or Qiaquick)

4) Check 1-2 l on a gel alongside the insert(s) that you will be cloning.

5) NEXT:

A) If you are cloning inserts that have no 5’ PO₄, go directly to cloning.

B) If desired, you can phosphatase-treat the vector to reduce background. (Use SAP = shrimp alkaline phosphatase, or CIP=calf intestinal alk. phosphatase; they differ wrt time & temp required to inactivate them.)

NOTES:

If you are planning to phosphatase your vector, it is best to do it immediately after restriction digest; I prefer SAP over CIP and SAP functions well in most restriction enzyme buffers.

If you are cloning PCR product staight from the PCR reaction, remember that synthetic oligos do not have 5’ PO₄, therefore you cannot use a phosphatased vector for cloning. In this case you will either have to use Blue/White selection or add PO₄ to the PCR product with polynucleotide kinase. (Alternatively, you may want to consider using a “T-tailed cloning vector--see _______.)