AlanFung:LabNotes/Capturing/2014-1-2: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
(13 intermediate revisions by the same user not shown) | |||
Line 56: | Line 56: | ||
*72C 3min | *72C 3min | ||
*Hold 15C | *Hold 15C | ||
==qPCR results== | ==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== | ==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" | | |||
|- 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" | | |||
|- style="font-size:12pt" valign="bottom" | |||
|style="font-weight:bold" height="15" | Total | |||
| align="center" | 100 | |||
| align="center" | | |||
| align="center" | | |||
|} | |||
*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]
- 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]
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