Matt:LabNotes/2015-3-25: Difference between revisions

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*Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
*Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
**Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
**Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
*Elute with 540ul H2O
*Elute with 540ul Elution buffer
**Should elute with < 400ul next time because it won't all fit in column
**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)
***Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
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~460ul of V4 probes: 71.3 ng/ul => 32.8ug
~460ul of V4 probes: 71.3 ng/ul => 32.8ug


===Repeat Production PCR===
*[[Matt:LabNotes/2015-1-5#Expansion_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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume (1X)'''
| align="center" style="background:#f0f0f0;"|'''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 | 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


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
*Divide into 9 pcr tubes of ~110ul with total amplicon of <10ug each
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 54: Line 84:


*Incubated at 37C for 1hr
*Incubated at 37C for 1hr
*Purified with 6 Zymo ssDNA/RNA columns
*Purified with 9 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 30ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**79.2ng/ul x 240ul = 19ug (76% yield)
**ng/ul x 270ul = ug (% yield)
 
<!--
===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====

Revision as of 20:47, 26 March 2015

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:032515 CA12kNov14 ProductionPCR V4.JPG

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

Lambda Exonuclease Digestion

  • 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 30ul each
  • Recombined and measured ssDNA with Nanodrop:
    • ng/ul x 270ul = ug (% yield)