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
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
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]