Matt Gethers/CRI, Thailand/Labwork/Creating Blunt Ends with Klenow
Creating Blunt Ends with Klenow
Note of warning: "The Klenow Fragment, exo-, is not recommended for fill-in reactions prior to DNA ligation, since it frequently adds one or more extra nucleotides to the 3’-terminus of a blunt-ended DNA substrate in a non-template directed fashion."
I'll see what protocol they normally use...
Pikoy normally uses End-IT DNA End-Repair Kit, so I may end up using that one.
Creating Blunt Ends using End-It
1. Make the following reaction mix:
| Reagent | Volume |
| DNA to repair | 10μl |
| TE | 24 μl |
| 10x repair buffer | 5 μl |
| 2.5 mM dNTP mix | 5 μl |
| 10 mm ATP | 5 μl |
| End-Repair enzyme mix | 1 μl |
2. Incubate at room temperature for 45 minutes.
3. Heat inactivate at 70oC for 10 minutes.
Run Notes
6.18.08
Made up 2x the volume indicated in the End-It protocol (not including the DNA to be repaired) and aliquotted to two tubes, one for the 1033/2724 amplicon and the second for pUC18 cut with SmaI. Added 10 μl of each as the protocol calls for. Incubated for 45 minutes at room temp, inactivated at ~70oC on heat block for 10 minutes, then in -20.
7.4.08
Made up the reaction in the protocol with 10 μl of pKn001.P1 cut with HincII. Started incubating at room temp at 1400; placed at 70oC at 1445 and removed from incubator at 1500.
7.10.08
Made up the reaction in the protocol with 10 μl of pKn001.P1 cut with HincII (7.10.08). Incubated at room temp for 45 minutes and then placed at 70oC for 10 minutes.
7.30.08
Used the kit to make blunt ends for BamHI digested pKn003, SphI digested pKn004, and NdeI and SacI digested Lox/Gentamicin cassette. For two vectors, I added ~15 μl of digest, 19 μl of water, and the other reagents as written. For the cassette, I added 35 μl of gel purification product, no water, and the other reagents as written. Incubated for 45 minutes at room temp and inactivated at 70 degrees for 10 minutes.
8.12.08
Used the kit to make blunt ends for 8.12.08 BamHI and XbaI digested pKn003 and 8.12.08 NdeI and SacI digested Lox/Gentamicin cassette. For the two vector digests, I added ~15 μl of digest, 19 μl of TE, and the other reagents as written. For the cassette, I added 35 μl of gel purification product, no water or TE, and the other reagents as written. Incubated for 45 minutes at room temp and inactivated at 70 degrees for 10 minutes.
