Matt:LabNotes/2015-3-25

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Revision as of 23:48, 26 March 2015 by >Mzcai (→‎Repeat Production PCR)
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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 Elution buffer
    • 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

Repeat Production PCR

  • 10nM 1st round amplicons
  • V4 master mix made in 1 15ml centrifugal tube
  • Poured into trough/reservoir and used multi-channel pipette to save time pipetting into pcr tubes
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: | 800px]]

Zymo DNA Clean & Concentrator 100

  • Tried using reservoir & multi-channel pipette removing product from 6 strips of pcr tubes but spilled ~1ml (product+binding buffer) pouring from reservoir into 50ml tubes
  • 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 400ul H2O
    • Somehow final volume is greater than 400ul
  • Measured concentration with Nanodrop:

~410ul of V4 probes: 76.1 ng/ul => 31.2ug

Potential Reasons for Low Yield

  • Second time I got 30ug when normal for EtOH precipitation method is 50ug
  • Possible reasons:
    • Potentially the vacuum is not strong enough to pull all solution through? (even though it's slow this is not likely)
    • Losing solution on the sides of the 50ml tubes used to mix binding buffer + amplicon (definitely happening but shouldn't be 40% of amplicon)
    • 400ul elution volume still too much to allow all elution buffer to get out of column membrane when centrifuged
  • From Alan: Try heating up elution buffer or incubating the column with elution buffer at 37C for 5min

Lambda Exonuclease Digestion

  • Combine 2 Production PCR products and Divide into 9 pcr tubes of ~110ul with total amplicon of <10ug each
Components Volume
Amplicon 96
10X Lambda Exo Buffer 12
Lambda Exonuclease 12
Total 120
  • Incubated at 37C for 1hr
  • Purified with 9 Zymo ssDNA/RNA columns
  • Eluted with 25ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 100.7ng/ul x 225ul = 22.7ug (70.9% yield)

Remove Amplification Adapters

USER

  • Split into 4 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 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)