AlanFung:LabNotes/Capturing/2014-1-2: Difference between revisions
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==PAGE Verification== | ==PAGE Verification== | ||
==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" | | |||
|} |
Revision as of 23:16, 2 January 2014
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 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
PCR amplification to check if capture works
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
PAGE Verification
PCR
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 |