Matt:LabNotes/2016-7-26

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Revision as of 22:20, 26 July 2016 by >Mzcai (→‎EtOH Precipitation)
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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: 313.6 ng/uL x 400uL = 125.44ug

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 2hr (normally 1hr)
  • Purified with 8 Zymo ssDNA/RNA columns
    • Dropped one column so only 7
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 118.7ng/ul x 280ul = 33.24ug

Remove Amplification Adapters

USER

  • Split into 5 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 56
USER 5
10X DpnII Buffer 8
H2O 11
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
    • 193.7 ng/ul x 100ul = 19.37 ug (% yield)

PAGE Size Selection

  • Run 4 gels
  • 200V for 40min
Components 4X Volume
V4 Probes 95
TBE-Urea Buffer 2X 95
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40

File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel1.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel2.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel3.jpg
File:2016-06-02 TB12kApr2016 V4 SizeSelect Gel4.jpg

EtOH Precipitation

  • Put cut out gel in 8 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
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
  • Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transferred spnt to fresh 1.5 mL tube (~3500ul)
  • Precipitated in 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 8 tubes at -80C for 30min
  • Spun 8 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
  • Discard supernatant and let dry in hood for 10min
  • Resuspend each tube with 10ul and combine

Qubit ssDNA

20.2ng/ul x 74ul = 1,494.8ng 20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM-->