Dinh 2011/NOTES/2011-9-2

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Revision as of 20:30, 2 September 2011 by >Dinh (Created page with '==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…')
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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)