Blunt-end Cloning of PCR Products

General Procedure =

PCR amplify your DNA fragment

Gel-Purify the PCR product

Blunt the fragment’s ends with T4 DNA polymerase

Clone the blunted PCR product into a blunted vector using “Blue-White” screening

1) Gel purify PCR product

2) If possible, estimate [DNA]

3) Blunt with T4 DNA polymerase (30 ml reaction):

DNA (1-10 mg)	_____ l
2mM dNTP	1.5l	Final=25uM each
10X T4 Polymerase Buffer	3l	contains BSA to give 50 ug/ml final
T4 DNA Pol.	1-5 units	stock usually 3 u/ul
H2O	QS 30l

37° C 5’

12° C 15’

75° C 10’

Phenol/Chloroform extract

Back-extract with 25-50l 10 mM Tris, pH 7.5

Final aqueous vol = _____

4) At this point, I like to desalt my DNA with a Qiaquick Spin column and elute in H2O. (Ethanol precipitation will work too.) For Qiaquick:

-add 5 vol of PB buffer

-check pH and adjust to pH < 7.5 if necessary with 3M NaOAc, pH5.2

-use column as per instructions in the manual

-elute DNA in volume of H2O to give approx. 50-200 ng/l

-the PCR fragment is now ready for blunt-end ligation.

NOTES:

At 37 deg the 3 to 5 exo is 3-fold stronger than the 5 to 3 polym. activity.

At 12 deg, the (ds 3 to 5 exo)/(5 to 3 pol.) ratio is closer to unity, so that the exo doesn’t overpower the polymerase. (Keeping all 4 dNTPs in the rxn also helps with this.)

Can also use Klenow. If you do,

BSA not necessary

dNTP also at 25 uM each

1 unit Klenow/ug DNA

Incubate at 25 deg for 15 min.

Stop with EDTA to 10 mM final and Heat at 75 deg for 10 min

Klenow is 50% active in all four NEB buffers

T4 DNA pol is active in all our NEB buffers + BSA, 100% active in NEB #3 + BSA

Klenow has a weaker ds 3 to 5 exonuclease activity than does T4 DNA pol.