Matt:LabNotes/2015-1-13: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
mNo edit summary
Line 104: Line 104:
*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
*Incubated at 37C for overnight (~15hrs)
<!--
 
===TBU Gel: Digestion Check===
===TBU Gel: Digestion Check===
*Combine into 1 tube for each set
*Combine into 1 tube
*2ul Digested Sample + 2ul 2X Urea Loading Dye
*2ul Sample + 2ul 2X Urea Loading Dye
*5ul Undigested sample (10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
[[File:2015-01-07_V4V7_Digest_GelCheck.jpg]]
[[File:]]
*Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters
*Gel ran poorly for some reason but I don't have anymore undigested probe to compare to (unless I want to waste 5ul of 10nM 1st rnd amplicon) so I will just check the gel when I do size selection
*To get a higher yield I continued digestion overnight
**The band to cut out should be 150bp so almost halfway between 200bp and 100bp ladder bands
 
===Continue Digestion===
*Add 100ul of the following to each tube
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X DpnII Buffer||10
|-
| USER||5
|-
| H2O||85
|-
| Total||100
|}
*Incubated at 37C for overnight (15 hrs)
 
===Zymo Column Purification===
===Zymo Column Purification===
*4 columns for each set
*Eluted 30ul each column (60ul total)
*Eluted 30ul each column (120ul total)
*Nanodrop
*Nanodrop
**V4: 45.0ng/ul x 120ul = 5.4ug
**41.7ng/ul x 60ul = 2.5ug (oddly low again like [[Matt:LabNotes/2015-1-5#Zymo_Column_Purification | last time]])
**V7: 22.8ng/ul x 120ul = 2.7ug (oddly low but checked with nanodrop twice)
***This time 33% yield which is better than 25% yield last time


===PAGE Size Selection===
===PAGE Size Selection===
*Run 2 gels for V4 and 1 gel for V7
*Run 1 gel for V7
**220V for 30min
**220V for 30min
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
| V4 Probes||120
|-
| TBE-Urea Buffer 2X||120
|}
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
|-
|-
| V7 Probes||120
| V7 Probes||60
|-
|-
| TBE-Urea Buffer 2X||120
| TBE-Urea Buffer 2X||60
|}
|}


Line 163: Line 135:
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| Low Mass Ladder||3
| Low Mass Ladder||1
|-
|-
| TBE-Urea Buffer 2X||15
| TBE-Urea Buffer 2X||5
|-
|-
| H2O||12
| H2O||4
|-
|-
| Total||30
| Total||10
|}
|}


*V4 Gel 1
[[File:]]
[[File:2015-01-08_SizeSelect_V4_Gel1.jpg]]
*V4 Gel 2
[[File:2015-01-08_SizeSelect_V4_Gel2.jpg]]
*V7 Gel 1
**Had trouble seeing blade and may have cut some extra above where I should have. However a mitigating factor is that V7 was digested much more completely than V4 based on intensity of band before cutting
[[File:2015-01-08_SizeSelect_V7_Gel1.jpg]]


===EtOH Precipitation===
===EtOH Precipitation===
Line 186: Line 152:
*Vortexed for 60 min at 37 C in incubator
*Vortexed for 60 min at 37 C in incubator
*Centrifuged at 15,000 rpm for 3 min at RT
*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 clear spnt. to 2 Nanosep columns 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 the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
*Transfered spnt to fresh 1.5 mL tube (~400ul per tube)
*Transfered spnt to 2 fresh 1.5 mL tube (~400ul per tube)
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Vortexed and placed the 6 tubes at -80C for overnight
*Vortexed and placed the 2 tubes at -80C for overnight<!--
*Spun 6 tubes at 10,000rpm at 4C for 30min
*Spun 6 tubes at 10,000rpm at 4C for 30min
*Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min
*Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min

Revision as of 02:49, 15 January 2015

CA12k_Nov2014_V7 Probe Production

Production PCR

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 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:011214 CA12kNov14 ProductionPCR V7.JPG

EtOH Precipitation

  • 6 15-ml tubes (with 8 wells of PCR product each) 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 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 6 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

  • Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~290ul of V7 probes: 93.2 ng/ul => ~27 ug

Lambda Exonuclease Digestion

  • Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
Components Volume
Amplicon 100
10X Lambda Exo Buffer 12.5
Lambda Exonuclease 12.5
Total 125
  • Incubated at 37C for 1hr
  • Purified with 3 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 62.9ng/ul x 120ul = 7.5ug (56% yield)

Remove Amplification Adapters

USER

  • Save 2ul for gel check later
  • Split into 2 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 59
USER 5
10X DpnII Buffer 8
H2O 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_V7 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)

TBU Gel: Digestion Check

  • Combine into 1 tube
  • 2ul Sample + 2ul 2X Urea Loading Dye
  • 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye

[[File:]]

  • Gel ran poorly for some reason but I don't have anymore undigested probe to compare to (unless I want to waste 5ul of 10nM 1st rnd amplicon) so I will just check the gel when I do size selection
    • The band to cut out should be 150bp so almost halfway between 200bp and 100bp ladder bands

Zymo Column Purification

  • Eluted 30ul each column (60ul total)
  • Nanodrop
    • 41.7ng/ul x 60ul = 2.5ug (oddly low again like last time)
      • This time 33% yield which is better than 25% yield last time

PAGE Size Selection

  • Run 1 gel for V7
    • 220V for 30min
Components 1X Volume
V7 Probes 60
TBE-Urea Buffer 2X 60
Components Volume
Low Mass Ladder 1
TBE-Urea Buffer 2X 5
H2O 4
Total 10

[[File:]]

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 2 Nanosep columns 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 2 fresh 1.5 mL tube (~400ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 2 tubes at -80C for overnight