Matt:LabNotes/2015-3-25: Difference between revisions

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**Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
**Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
*Elute with 540ul H2O
*Elute with 540ul H2O
**Should elute with < 400ul next time because it won't all fit in column
***Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:
~540ul of V4 probes: __ ng/ul => ug
~460ul of V4 probes: 71.3 ng/ul => 32.8ug
 
 
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===Qia Column Purification===
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
*Eluted each column with 50ul and combined all into one 1.5ml
*Measured concentration with Nanodrop:
~575ul of V4 probes: 86.9 ng/ul => ~50 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===

Revision as of 23:06, 25 March 2015

CA12k_Nov2014_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4900
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:032515 CA12kNov14 ProductionPCR V4.JPG

Zymo DNA Clean & Concentrator 100

  • Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
    • Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
  • Elute with 540ul H2O
    • Should elute with < 400ul next time because it won't all fit in column
      • Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
  • Measured concentration with Nanodrop:

~460ul of V4 probes: 71.3 ng/ul => 32.8ug