Revision as of 00:22, 25 June 2016 by >Mzcai
TB12k_Apr2016 V7 Mouse Brain Probe Preparation[edit]
Expansion PCR[edit]
Component
|
Volume
|
Seed oligo (100X Dilution) |
15
|
F/R V7 Primer Mix (10uM) |
12
|
2X Kapa MM |
75
|
H2O |
48
|
Total |
150
|
Program
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold
File:2016-06-20 TB12kApr2016 V7 ExpansionPCR.jpg
- Purified with 3 Qiagen PCR columns (50ul PCR product each)
- Quantify with Qubit dsDNA:
- 9.2ng/ul / (195bp*607.4Da/bp+157.9Da) = 77.57 nmol/L
- Dilute to 10nM
- 77.57nM x 144ul = 10nM x 1117ul
- Add 973ul H2O
Production PCR[edit]
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon TB12k_Apr2016_V7 (10nM) |
0.2 |
10
|
2X KAPA SYBG MM |
50 |
2500
|
AP1V7U (100uM) |
0.4 |
20
|
AP2V7 (100uM) |
0.4 |
20
|
H2O |
49 |
2450
|
Total |
100 |
5000
|
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
File:2016-06-20 TB12kApr2016 V7 ProductionPCR.jpg
EtOH Precipitation[edit]
- 4 5-ml tubes (with 24 wells (3 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]
- 4 columns elute 50ul each
- Nanodrop: 193.0 ng/uL x 200uL = 38.6ug
Lambda Exo[edit]
- Divide into 4 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 1.5 hr (normally 1 hr)
- Purified with 4 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 68.4ng/ul x 160ul = 10.944 ug (% yield)
Remove Amplification Adapters[edit]
- Split into 3 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
53
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
14
|
Total |
80
|
- Incubate at 37C for 2.5 hours
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
2
|
100uM RE-DpnII_V7 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 (60ul total)
- Nanodrop
- 111.4ng/ul x 60ul = 6.684ug (62.5% yield)
PAGE Size Selection[edit]
- Run 2 gels
- 200V for 40min
Components
|
2X Volume
|
V4 Probes |
60
|
TBE-Urea Buffer 2X |
60
|
Components
|
2X Volume
|
Low Mass Ladder |
2
|
TBE-Urea Buffer 2X |
10
|
H2O |
8
|
Total |
20
|
File:2016-06-23 TB12kApr2016V7 ProbeProduction SizeSelect.jpg
- Only saw one faint band above main band that was cut out
- That means either the USER or DpnII digestion was very complete or did not occur at all
- I am sure I used the corrent DpnII-V7 oligo
- Can run a gel comparing with 1st round amplicons to check for size next time
EtOH Precipitation[edit]
- Put cut out gel in 4 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 4 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 450ul spnt to fresh 1.5 mL tube
- Precipitated in 4 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C for 30min
- Spun 4 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]
18.2ng/ul x 40ul = 728ng
18.2ng/ul / (160nt x 303.7Da/nt + 79Da) = 374 nM