Matt:LabNotes/2016-11-10: Difference between revisions

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>Mzcai
Line 101: Line 101:
*Incubated at 94C for 2 min, then 37C for 3 min
*Incubated at 94C for 2 min, then 37C for 3 min
*Added 5ul DpnII
*Added 5ul DpnII
*Incubated at 37C for overnight (~15hrs)
*Incubated at 37C for overnight (~12hrs)
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===Zymo Column Purification===
===Zymo Column Purification===
*Eluted 20ul each column (180ul total)
*Eluted 20ul each column (180ul total)
*Nanodrop
*Nanodrop
**87.3 ng/ul x 60ul = 5.2ug (34% yield lower than normal)
**80.1 ng/ul x 180ul = 14.4ug (34% yield lower than normal)
 
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===Gel Check===
*Pre-run 12 well TBU gel for 20min at 250V
*Rinse wells
#Ladder
#*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X TBU Loading Dye
#User/DpnII-Digested
#*2ul Sample + 2ul 2X TBU Loading Dye
#Lambda-Digested
#*2ul Sample + 2ul 2X TBU Loading Dye
*Pre-heat samples to 75C for 10min and then put on ice
*Load samples in middle lanes of gel
*Run for 33min at 210V
[[File:2016-11-08_CA12kOct2016_V4_GelCheck.jpg|250px]]
 
===PAGE Size Selection===
===PAGE Size Selection===
*Run 2 gels
*Run 2 gels

Revision as of 20:25, 14 November 2016

CA12k_Oct2016 V4 Probe Preparation

Production PCR

  • V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Oct2016_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
  • Did one plate - 96 wells 100ul each

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold 450px

EtOH Precipitation

  • 16 5-ml tubes each with
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • 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 16 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

  • 16 columns elute 50ul each
  • Nanodrop: 170.3 ng/uL x 800uL = 136.24ug

Lambda Exo

  • Divide into 14 pcr tubes of 100ul (ideally should be <10ug each)
Components Volume
Amplicon 57
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 23
Total 100
  • Incubated at 37C for 1hr
  • Purified with 14 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 72.4ng/ul x 560ul = 40.54ug (60% yield)

Remove Amplification Adapters

USER

  • Split into 9 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 62.2
USER 5
10X DpnII Buffer 8
H2O 4.8
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 (~12hrs)

Zymo Column Purification

  • Eluted 20ul each column (180ul total)
  • Nanodrop
    • 80.1 ng/ul x 180ul = 14.4ug (34% yield lower than normal)