Matt:LabNotes/2016-7-26

From ZhangLabWiki
Revision as of 23:52, 28 July 2016 by >Mzcai (→‎EtOH Precipitation)
Jump to navigation Jump to search

TB12k_Apr2016 V4 Probe Preparation

Production PCR

  • Make 10ml V4 master mix for 96 wells
Components Volume (1X) Volume (100X)
First round amplicon TB12k_Apr2016_V4 (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 10,000

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold

  • Did in regular thermocyclers

EtOH Precipitation

  • 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 30 min
  • Centrifuged at 3000rpm at 4C for 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • 8 columns elute 50ul each
  • Nanodrop: 374.4 ng/uL x 400uL = 149.76ug

Lambda Exo

  • Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 3hr (normally 1hr)
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 167.0ng/ul x 320ul = 53.44ug

Remove Amplification Adapters

USER

  • Split into 6 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 53.3
USER 5
10X DpnII Buffer 8
H2O 13.7
Total 80
  • Incubate at 37C for 2.5 hours

DpnII

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 2
100uM RE-DpnII_V4 guide oligo 5
H2O 8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (~15hrs)

Zymo Column Purification

  • Eluted 20ul each column (100ul total)
  • Nanodrop
    • 229.0 ng/ul x 140ul = 32.06 ug (59% yield)

PAGE Size Selection

  • Run 9 gels (wanted to do 10 but not enough gels)
  • 200V for 40min
Components 9X Volume
V4 Probes 135
TBE-Urea Buffer 2X 135
Components 9X Volume
Low Mass Ladder 9
TBE-Urea Buffer 2X 45
H2O 36
Total 90

EtOH Precipitation

  • Put cut out gel in 18 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
  • Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
  • Added 450 ul of 1X TE buffer to each
  • Vortexed for 60min at 37 C in incubator