Matt:LabNotes/2016-9-14: Difference between revisions
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>Mzcai m (→Production PCR) |
>Mzcai |
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(9 intermediate revisions by the same user not shown) | |||
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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
Line 45: | Line 43: | ||
*Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another | *Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another | ||
*Measured concentration with Nanodrop: | *Measured concentration with Nanodrop: | ||
~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug | |||
~600ul of V6 probes: | |||
===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide each into | *Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| Amplicon|| | | Amplicon||75 | ||
|- | |- | ||
| 10X Lambda Exo Buffer||10 | | 10X Lambda Exo Buffer||10 | ||
Line 60: | Line 57: | ||
| Lambda Exonuclease||10 | | Lambda Exonuclease||10 | ||
|- | |- | ||
| H2O|| | | H2O||5 | ||
|- | |- | ||
| Total||100 | | Total||100 | ||
Line 66: | Line 63: | ||
*Incubated at 37C for 1hr | *Incubated at 37C for 1hr | ||
*Purified with | *Purified with 8 Zymo ssDNA/RNA columns | ||
*Eluted with 40ul each | *Eluted with 40ul each | ||
*Recombined and measured ssDNA with Nanodrop: | *Recombined and measured ssDNA with Nanodrop: | ||
**V6: | **V6: 80.3ng/ul x 320ul = 25.7ug (75.6% yield) | ||
===Remove Amplification Adapters=== | ===Remove Amplification Adapters=== | ||
====USER==== | ====USER==== | ||
*Split each set into | *Split each set into 5 PCR tubes and add 5ul USER | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| SS-amplicon|| | | SS-amplicon||63 | ||
|- | |- | ||
| USER||5 | | USER||5 | ||
|- | |- | ||
| Total|| | | DpnII 10X||8 | ||
|- | |||
| H2O||4 | |||
|- | |||
| Total||80 | |||
|} | |} | ||
*Incubate at 37C for 3 hours | *Incubate at 37C for 3 hours | ||
====DpnII==== | ====DpnII==== | ||
*Added 15ul of the following | *Added 15ul of the following | ||
Line 91: | Line 93: | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| 10X DpnII Buffer|| | | 10X DpnII Buffer||2 | ||
|- | |||
| 100uM RE-DpnII_V6 guide oligo||5 | |||
|- | |||
| H2O||8 | |||
|- | |- | ||
|} | |} | ||
*Incubated at 94C for 2 min, then 37C for 3 min | *Incubated at 94C for 2 min, then 37C for 3 min | ||
*Added 5ul DpnII | *Added 5ul DpnII | ||
*Incubated at 37C for overnight ( | *Incubated at 37C for overnight (12 hrs) | ||
===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*Eluted 20ul each column (100ul total) | |||
*Eluted 20ul each column ( | |||
*Nanodrop | *Nanodrop | ||
**V6: | **V6: 118.9ng/ul x 100ul = 11.9ug | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
Line 119: | Line 115: | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''4X Volume''' | ||
|- | |- | ||
| V6 Probes|| | | V6 Probes||100 | ||
|- | |- | ||
| H2O || | | H2O||20 | ||
|- | |- | ||
| TBE-Urea Buffer 2X||120 | | TBE-Urea Buffer 2X||120 | ||
Line 142: | Line 138: | ||
===EtOH Precipitation=== | ===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. | *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.) | ||
*Transfered the gel remaining in 0.5 mL tube to | *Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip. | ||
*Added | *Added 450 ul of 1X TE buffer | ||
*Vortexed for 60 min at 37 C in incubator | *Vortexed for 60 min at 37 C in incubator | ||
*Centrifuged at 15,000 rpm for 3 min at RT | *Centrifuged at 15,000 rpm for 3 min at RT | ||
Line 155: | Line 151: | ||
*Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min | *Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min | ||
*Discard supernatant and let dry in hood for 10min | *Discard supernatant and let dry in hood for 10min | ||
*Resuspend each tube with 10ul and recombine ( | *Resuspend each tube with 10ul and recombine (80ul total) | ||
*Measure with qubit | |||
*V6: 13.1 ng/ul => 13.1 ng/ul / (150nt*325Da/nt + 79Da) = 268.2nM (80ul) | |||
*V6: | |||
Latest revision as of 22:20, 21 September 2016
CA12k_Nov2014 V6 (RevComp of V4) Probe Production[edit]
Production PCR[edit]
- V6 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V6 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V6U (100uM) | 0.4 | 20 |
AP2V6 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
EtOH Precipitation[edit]
- 12 5-ml tubes (with 8 wells of PCR product each)
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 1hr
- 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 12 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]
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
- Measured concentration with Nanodrop:
~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug
Lambda Exonuclease Digestion[edit]
- Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 75 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 5 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V6: 80.3ng/ul x 320ul = 25.7ug (75.6% yield)
Remove Amplification Adapters[edit]
USER[edit]
- Split each set into 5 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 63 |
USER | 5 |
DpnII 10X | 8 |
H2O | 4 |
Total | 80 |
- Incubate at 37C for 3 hours
DpnII[edit]
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 2 |
100uM RE-DpnII_V6 guide oligo | 5 |
H2O | 8 |
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (12 hrs)
Zymo Column Purification[edit]
- Eluted 20ul each column (100ul total)
- Nanodrop
- V6: 118.9ng/ul x 100ul = 11.9ug
PAGE Size Selection[edit]
- Run 4 gels for V6
- 200V for 35min
Components | 4X Volume |
V6 Probes | 100 |
H2O | 20 |
TBE-Urea Buffer 2X | 120 |
Components | Volume |
Low Mass Ladder | 4 |
TBE-Urea Buffer 2X | 20 |
H2O | 16 |
Total | 40 |
EtOH Precipitation[edit]
- 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.)
- Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer
- Vortexed for 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to 8 Nanosep column and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to 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 30 min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 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 recombine (80ul total)
- Measure with qubit
- V6: 13.1 ng/ul => 13.1 ng/ul / (150nt*325Da/nt + 79Da) = 268.2nM (80ul)