Dinh 2011/NOTES/2011-9-2
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T4 Polynucleotide Kinase
- NEB kit:
T4 PNK (10U/ul) PNK buffer (10x)
- Up 300 pmol of 5' termini in a 50 ul reaction containing 1X buffer and 10 Units of T4 PNK. The enzyme has 100% activity in T4 DNA Ligase (Quick Ligase buffer).
Phosphorylating the splint:
APV6_Splint (100uM) 3ul (300pmol) T4 PNK (10U/ul) 1ul (10U) H2O 2.5ul QuickLigase Buffer (2x) 6.5ul (1x) ============================================ 13ul
- Need 330,000x25,000 molecules = 8.25 x 10^9 molecules of probes
1.25 pmoles of probes will give ~90x of this required amount.
Phosphorylating the probes:
98ng/ul lambda exo digested probes, 2.15uM 6ul (12.9pmol) T4 PNK (10U/ul) 0.5ul (5U) QuickLigase Buffer (2x) 6.5ul (1x) ====================================================================== 13ul
- Purify both with 1.8x AMPure beads (24 ul)