Revision as of 22:01, 28 March 2015 by >Mzcai
CA12k_Nov2014_V4 Probe Production
Production PCR
Components
|
Volume (1X)
|
Volume (100X)
|
First round amplicon CA12k_Nov14_V7 (10nM) |
0.2 |
20
|
2X KAPA SYBG MM |
50 |
5000
|
AP1V4U (100uM) |
0.4 |
40
|
AP2V4 (100uM) |
0.4 |
40
|
H2O |
49 |
4900
|
Total |
100 |
10000
|
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
File:032515 CA12kNov14 ProductionPCR V4.JPG
Zymo DNA Clean & Concentrator 100
- Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
- Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
- Elute with 540ul Elution buffer
- Should elute with < 400ul next time because it won't all fit in column
- Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
- Measured concentration with Nanodrop:
~460ul of V4 probes: 71.3 ng/ul => 32.8ug
Repeat Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 1 15ml centrifugal tube
- Poured into trough/reservoir and used multi-channel pipette to save time pipetting into pcr tubes
Components
|
Volume (1X)
|
Volume (100X)
|
First round amplicon CA12k_Nov14_V7 (10nM) |
0.2 |
20
|
2X KAPA SYBG MM |
50 |
5000
|
AP1V4U (100uM) |
0.4 |
40
|
AP2V4 (100uM) |
0.4 |
40
|
H2O |
49 |
4900
|
Total |
100 |
10000
|
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
File:032615 CA12kNov14 ProductionPCR V4.JPG
Zymo DNA Clean & Concentrator 100
- Tried using reservoir & multi-channel pipette removing product from 6 strips of pcr tubes but spilled ~1ml (product+binding buffer) pouring from reservoir into 50ml tubes
- Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
- Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
- Elute with 400ul H2O
- Somehow final volume is greater than 400ul
- Measured concentration with Nanodrop:
~410ul of V4 probes: 76.1 ng/ul => 31.2ug
Potential Reasons for Low Yield
- Second time I got 30ug when normal for EtOH precipitation method is 50ug
- Possible reasons:
- Potentially the vacuum is not strong enough to pull all solution through? (even though it's slow this is not likely)
- Losing solution on the sides of the 50ml tubes used to mix binding buffer + amplicon (definitely happening but shouldn't be 40% of amplicon)
- 400ul elution volume still too much to allow all elution buffer to get out of column membrane when centrifuged
- From Alan: Try heating up elution buffer or incubating the column with elution buffer at 37C for 5min
Lambda Exonuclease Digestion
- Combine 2 Production PCR products and Divide into 9 pcr tubes of ~110ul with total amplicon of <10ug each
Components
|
Volume
|
Amplicon |
96
|
10X Lambda Exo Buffer |
12
|
Lambda Exonuclease |
12
|
Total |
120
|
- Incubated at 37C for 1hr
- Purified with 9 Zymo ssDNA/RNA columns
- Eluted with 25ul each
- Recombined and measured ssDNA with Nanodrop:
- 100.7ng/ul x 225ul = 22.7ug (70.9% yield)
Remove Amplification Adapters
USER
- Split into 4 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
56
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
11
|
Total |
80
|
- Incubate at 37C for 2 hours
DpnII
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
2
|
100uM RE-DpnII_V4 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)
Zymo Column Purification
- Eluted 30ul each column (115ul total)
- Nanodrop
- 126.8ng/ul x 115ul = 14.6ug (66% yield)
PAGE Size Selection
Components
|
4X Volume
|
V4 Probes |
115
|
TBE-Urea Buffer 2X |
115
|
Components
|
4X Volume
|
Low Mass Ladder |
4
|
TBE-Urea Buffer 2X |
20
|
H2O |
16
|
Total |
40
|
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.
- Transferred 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 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 8 Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to 8 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 8 tubes at -80C for 20min