Ligations and Repair

Ligations and Repair

Sticky-end Ligation

Digest plasmids containing vector and insert fragments with appropriate enzymes.Purify vector and insert fragments by appropriate method. Resuspend DNA in 20 ul TE buffer. Estimate DNA concentration by agarose gel electrophoresis. Do not ligate >500ng total.

x ul vector (use molar ratio of 2:1 Insert to Vector)

y ul insert

2 ul 10 X ligation buffer

1 ul 200 mM DTT

1 ul 1 mM ATP

1 ul 1 mg/ml BSA

14.5 ul - (x + y ul) dd water

0.5 ul T4 DNA ligase (use units of ligase as suggested by manufacturer)

Incubate at 14 degrees C for 4 hours. Inactivate enzyme at 65 degrees C for 10 min.

 

Blunt-end Ligation

x ul vector (use molar ratio of 2:1 Insert to Vector)

y ul insert

2 ul 10 X ligation buffer

1 ul 200 mM DTT

1 ul 20 mM ATP

1 ul 1 mg/ml BSA

14.5 ul - (x + y ul) dd water

1 ul T4 DNA ligase (use units of ligase as suggested by manufacturer)

Incubate at 14 degrees C for overnight. Inactivate enzyme at 65 degrees C for 10 min.

Transform E coli with 2-5 ul ligation mix

10X Ligation buffer

500 mM Tris-HCL

100 mM MgCl2; pH 7.5.

 

Digestion and T4 Polymerase Repair

Digest DNA in O'Farrell buffer (50 ul reaction volume).

Use 1 ul for gel electrophoresis to check for completion of digestion.

49 ul DNA

5 ul 10 X O'Farrell buffer

5 ul 10 mM DTT

2.5 ul 1 mg/ml BSA

2.5 ul 10 mM dNTP's

34 ul ddwater

1 ul T4 DNA polymerase

Incubate at Room Temperature for 15 min

Inactivate at 65 degrees C for 10 min

-- To dephosphorylate add 2 ul of 10 mM DTT and 1 ul of CIAP (37 degrees C for 1 hr)

 

10X O'Farell Buffer

0.33M Tris-HAc pH 8.22

0.66M KOAc

0.1M MgOAc