Matt:LabNotes/2015-1-15

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Revision as of 03:09, 21 January 2015 by >Mzcai (→‎EtOH Precipitation)
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CA12k_Nov2014 V4 and V7 Probe Production

Production PCR

Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V4 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V4U (100uM) 0.4 20
AP2V4 (100uM) 0.4 20
H2O 49 2450
Total 100 5000
  • V7 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V7U (100uM) 0.4 20
AP2V7 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 15sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]

EtOH Precipitation

  • 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 1hr
  • 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 12 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 50ul H2O

Qia Column Purification

  • Purified in 6 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another
  • Measured concentration with Nanodrop:

~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug

Lambda Exonuclease Digestion

  • Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 1hr
  • Purified with 3 Zymo ssDNA/RNA columns each
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • V4: 77.0ng/ul x 120ul = 9.24ug (78.6% yield)
    • V7: 61.0ng/ul x 120ul = 7.32ug (59.8% yield)

Remove Amplification Adapters

USER

  • Split each set into 2 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 60
USER 5
H2O 20
Total 85
  • Incubate at 37C for 2.5 hours

DpnII

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 10
100uM RE-DpnII guide oligo 5
 RE-DpnII guide oligo:
 V4: RE DpnII V4
 V7: RE DpnII V7
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight

TBU Gel: Digestion Check

  • Combine into 1 tube for each set
  • 2ul Digested Sample + 2ul 2X Urea Loading Dye
  • 5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
  • 2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye)

File:2015-01-20 V4V7 Digest GelCheck.jpg

  • V7 Undigested (middle lane) is extremely faint but can see band around 200bp
  • Not sure why V4 lane is squiggly but can see the 3 bands
  • V7 lane shows only 2 bands but both are below the undigested V7 band

Zymo Column Purification

  • 2 columns for each set
  • Eluted 30ul each column (60ul total)
  • Nanodrop
    • V4: 78.8ng/ul x 60ul = 4.7ug
    • V7: 38.8ng/ul x 60ul = 2.3ug

PAGE Size Selection

  • Run 2 gels for V4 and 1 gel for V7
    • 200V for 30min
Components 2X Volume
V4 Probes 60
TBE-Urea Buffer 2X 60
Components 1X Volume
V7 Probes 60
TBE-Urea Buffer 2X 60
Components Volume
Low Mass Ladder 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • V4 Gel 1

File:2015-01-20 SizeSelect V4 Gel1.jpg

  • V4 Gel 2

File:2015-01-20 SizeSelect V4 Gel2.jpg

  • V7 Gel 1

File:2015-01-20 SizeSelect V7 Gel1.jpg

EtOH Precipitation

  • Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
  • Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
  • Added 900 ul of 1X TE buffer
  • Vortexed for 60 min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to 6 Nanosep column and centrifuged at 15,000 rpm for 3 min
  • Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transfered spnt to fresh 1.5 mL tube (~450ul per tube except for 1 V4 tube which spilled)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 6 tubes at -80C for overnight