Revision as of 19:01, 2 August 2016 by >Mzcai
TB12k_Apr2016 V4 Probe Preparation[edit]
Production PCR[edit]
- Make 10ml V4 master mix for 96 wells
Components
|
Volume (1X)
|
Volume (100X)
|
First round amplicon TB12k_Apr2016_V4 (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 |
10,000
|
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold
- Did in regular thermocyclers
EtOH Precipitation[edit]
- 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30 min
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification[edit]
- 8 columns elute 50ul each
- Nanodrop: 374.4 ng/uL x 400uL = 149.76ug
Lambda Exo[edit]
- Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components
|
Volume
|
Amplicon |
50
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
30
|
Total |
100
|
- Incubated at 37C for 3hr (normally 1hr)
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 167.0ng/ul x 320ul = 53.44ug
Remove Amplification Adapters[edit]
- Split into 6 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
53.3
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
13.7
|
Total |
80
|
- Incubate at 37C for 2.5 hours
- 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[edit]
- Eluted 20ul each column (100ul total)
- Nanodrop
- 229.0 ng/ul x 140ul = 32.06 ug (59% yield)
PAGE Size Selection[edit]
File:2016-07-28 TB12kApr2016 V4 ProbeProduction GelCheck.jpg
- You can see the 3 bands at the top
- The full digested band is the brightest (third from top) and also kind of blurry due to amount
- Run 6 gels for 6/7/2016 batch
Components
|
6X Volume
|
V4 Probes |
95
|
TBE-Urea Buffer 2X |
95
|
- Run 9 gels for this batch (wanted to do 10 but not enough gels)
Components
|
9X Volume
|
V4 Probes |
135
|
TBE-Urea Buffer 2X |
135
|
Components
|
15X Volume
|
Low Mass Ladder |
15
|
TBE-Urea Buffer 2X |
75
|
H2O |
60
|
Total |
150
|
- 200V for 40min
- No gel images because too many gels
- Bands looked good, could see very faint band of ~200bp and a less faint band below that and finally the completely digested probe band was brightest below that
EtOH Precipitation[edit]
- Put cut out gel in 30 0.5mL tubes (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 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer to each
- Vortexed for 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 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 fresh 1.5 mL tube
- Precipitated in 30 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 30 tubes at -80C for over the weekend
- Spun 30 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul and combine
Qubit ssDNA[edit]
22.7ng/ul x 300ul = 6,810ng
22.7ng/ul / (155nt x 303.7Da/nt + 79Da) = 478nM