AlanFung:LabNotes/Capturing/2014-1-2: Difference between revisions

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(Created page with "==Helping Dheeraj to test Klentaq capture reactions== *Dheeraj reached out to me saying he wants to start a qPCR reaction on the Klentaq capture reaction, he was working with ...")
 
>Alan6017518
 
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*Dheeraj reached out to me saying he wants to start a qPCR reaction on the Klentaq capture reaction, he was working with Noi to test the protocol, but Noi is sick and I will be helping to perform the qPCR
*Dheeraj reached out to me saying he wants to start a qPCR reaction on the Klentaq capture reaction, he was working with Noi to test the protocol, but Noi is sick and I will be helping to perform the qPCR
*They have 12 samples and we will be amplifying the captured samples with qPCR using the same protocol as it in Stoffol capture
*They have 12 samples and we will be amplifying the captured samples with qPCR using the same protocol as it in Stoffol capture
*1-6 done in condition 16 hours incubation 4 hours polymerization
*the second set we did the same 20 hours incubation and 20 hours polymerization
==Protocol==
==Protocol==
==PCR amplification to check if capture works==
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="font-size:12pt;font-weight:bold"  valign="bottom"
| width="137" height="15" | Components
| width="65" | 1x rxn (ul)
| width="65" | 12x rxn (ul)
| width="65" | Final Conc.
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | Circularized DNA template
| align="center" | 2.5
| align="center" | 0
| align="center" | &nbsp;
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | KAPA SSYBR FAST MM 2X
| align="center" | 12.5
| align="center" | 150
| 1X
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | 10uM AmpF6.4Sol
| align="center" | 0.5
| align="center" | 6
| 200nM
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | 10uM AmpR6.3Ind23
| align="center" | 0.5
| align="center" | 6
| 200nM
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | H2O
| align="center" | 10
| align="center" | 120
| align="center" | &nbsp;
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | Total
| align="center" | 25
| align="center" | &nbsp;
| align="center" | &nbsp;
|}
*98C 30sec
*(98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles
*(98C 10sec -> 72C 20sec) X 10-15 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau)
*72C 3min
*Hold 15C
==qPCR results==
[[File:01022014_1.JPG|400px|16Hr,4Hr]]
[[File:01022014_2.JPG|400px|20Hr,20Hr]]
*L:16Hr, 4Hr R:20Hr, 20Hr
*For 16Hr,4Hr set we should stop at 8 cycles
*For 20Hr, 20Hr set we should stop the amplification after 7 cycles
==PAGE Verification==
[[File:ZhangLab_2 2014-01-02 15hr 41min.jpg|400px|PAGE]]
==PCR==
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="font-size:12pt;font-weight:bold"  valign="bottom"
| width="137" height="15" | Components
| width="65" | 1x rxn (ul)
| width="65" | 12x rxn (ul)
| width="65" | Final Conc.
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | Circularized DNA template
| align="center" | 10
| align="center" | 0
| align="center" | &nbsp;
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | KAPA SSYBR FAST MM 2X
| align="center" | 50
| align="center" | 600
| 1X
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | 10uM AmpF6.4Sol
| align="center" | 2
| align="center" | 24
| 200nM
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | 10uM AmpR6.3IndX
| align="center" | 2
| align="center" | 0
| 200nM
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | H2O
| align="center" | 36
| align="center" | 432
| align="center" | &nbsp;
|- style="font-size:12pt"  valign="bottom"
|style="font-weight:bold" height="15" | Total
| align="center" | 100
| align="center" | &nbsp;
| align="center" | &nbsp;
|}
*98C 30sec
*(98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles
*(98C 10sec -> 72C 20sec) X 10 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau)
*72C 3min
*Hold 15C
*Used Index #1-12 for amplification
[[File:01022014_16hr_4hr.JPG|400px]]
[[File:01022014_20hr_20hr.JPG|400px]]
*L:16Hr, 4Hr R:20Hr, 20Hr
==AMPure bead purification protocol==
*Mix 1 volume of bead with PCR product by pipetting 10X
*Leave at RT for 8min
*Sit on magnetic 96-ring stand for 5min
*Wash twice with freshly prepared 200ul 80% EtOH (after adding 80% EtOH, wait for 30sec before pipette up and down few times and discard spnt, and avoid to touch the beads)
*Dry the beads for 3 min (do not overdry)
*Resuspend the bead with 50ul EB buffer
*Leave at RT for 2 min
*Sit on magnetic 96-ring stand for 3-5min
*Transfer elute DNA to new tube
==PAGE Gel Size Selection==

Latest revision as of 19:32, 3 January 2014

Helping Dheeraj to test Klentaq capture reactions[edit]

  • Dheeraj reached out to me saying he wants to start a qPCR reaction on the Klentaq capture reaction, he was working with Noi to test the protocol, but Noi is sick and I will be helping to perform the qPCR
  • They have 12 samples and we will be amplifying the captured samples with qPCR using the same protocol as it in Stoffol capture
  • 1-6 done in condition 16 hours incubation 4 hours polymerization
  • the second set we did the same 20 hours incubation and 20 hours polymerization

Protocol[edit]

PCR amplification to check if capture works[edit]

Components 1x rxn (ul) 12x rxn (ul) Final Conc.
Circularized DNA template 2.5 0  
KAPA SSYBR FAST MM 2X 12.5 150 1X
10uM AmpF6.4Sol 0.5 6 200nM
10uM AmpR6.3Ind23 0.5 6 200nM
H2O 10 120  
Total 25    
  • 98C 30sec
  • (98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles
  • (98C 10sec -> 72C 20sec) X 10-15 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau)
  • 72C 3min
  • Hold 15C

qPCR results[edit]

16Hr,4Hr 20Hr,20Hr

  • L:16Hr, 4Hr R:20Hr, 20Hr
  • For 16Hr,4Hr set we should stop at 8 cycles
  • For 20Hr, 20Hr set we should stop the amplification after 7 cycles

PAGE Verification[edit]

PAGE

PCR[edit]

Components 1x rxn (ul) 12x rxn (ul) Final Conc.
Circularized DNA template 10 0  
KAPA SSYBR FAST MM 2X 50 600 1X
10uM AmpF6.4Sol 2 24 200nM
10uM AmpR6.3IndX 2 0 200nM
H2O 36 432  
Total 100    
  • 98C 30sec
  • (98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles
  • (98C 10sec -> 72C 20sec) X 10 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau)
  • 72C 3min
  • Hold 15C
  • Used Index #1-12 for amplification

File:01022014 16hr 4hr.JPG File:01022014 20hr 20hr.JPG

  • L:16Hr, 4Hr R:20Hr, 20Hr

AMPure bead purification protocol[edit]

  • Mix 1 volume of bead with PCR product by pipetting 10X
  • Leave at RT for 8min
  • Sit on magnetic 96-ring stand for 5min
  • Wash twice with freshly prepared 200ul 80% EtOH (after adding 80% EtOH, wait for 30sec before pipette up and down few times and discard spnt, and avoid to touch the beads)
  • Dry the beads for 3 min (do not overdry)
  • Resuspend the bead with 50ul EB buffer
  • Leave at RT for 2 min
  • Sit on magnetic 96-ring stand for 3-5min
  • Transfer elute DNA to new tube

PAGE Gel Size Selection[edit]