Matt:LabNotes/2015-1-15: Difference between revisions
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>Mzcai |
>Mzcai |
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*Not sure why V4 lane is squiggly but can see the 3 bands | *Not sure why V4 lane is squiggly but can see the 3 bands | ||
*V7 lane shows only 2 bands but both are below the undigested V7 band | *V7 lane shows only 2 bands but both are below the undigested V7 band | ||
===Zymo Column Purification=== | ===Zymo Column Purification=== | ||
*2 columns for each set | *2 columns for each set | ||
*Eluted 30ul each column (60ul total) | *Eluted 30ul each column (60ul total) | ||
*Nanodrop | *Nanodrop | ||
**V4: | **V4: 78.8ng/ul x 60ul = 4.7ug | ||
**V7: | **V7: 38.8ng/ul x 60ul = 2.3ug | ||
===PAGE Size Selection=== | ===PAGE Size Selection=== | ||
*Run 2 gels for V4 and 1 gel for V7 | *Run 2 gels for V4 and 1 gel for V7 | ||
** | **200V for 30min | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 173: | Line 173: | ||
| Total||30 | | Total||30 | ||
|} | |} | ||
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*V4 Gel 1 | *V4 Gel 1 | ||
[[File:2015-01-08_SizeSelect_V4_Gel1.jpg]] | [[File:2015-01-08_SizeSelect_V4_Gel1.jpg]] |
Revision as of 22:51, 20 January 2015
CA12k_Nov2014 V4 and V7 Probe Production
- Repeat Matt:LabNotes/2015-1-5
Production PCR
- V4 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_V4 (10nM) | 0.2 | 10 |
2X KAPA SYBG MM | 50 | 2500 |
AP1V4U (100uM) | 0.4 | 20 |
AP2V4 (100uM) | 0.4 | 20 |
H2O | 49 | 2450 |
Total | 100 | 5000 |
- V7 master mix
Components | Volume (1X) | Volume (50X) |
First round amplicon CA12k_Nov14_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 15sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]
EtOH Precipitation
- 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
- 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 50ul H2O
Qia Column Purification
- Purified in 6 columns following Qiagen protocol
- Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another
- Measured concentration with Nanodrop:
~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug
Lambda Exonuclease Digestion
- Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each
Components | Volume |
Amplicon | 50 |
10X Lambda Exo Buffer | 10 |
Lambda Exonuclease | 10 |
H2O | 30 |
Total | 100 |
- Incubated at 37C for 1hr
- Purified with 3 Zymo ssDNA/RNA columns each
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V4: 77.0ng/ul x 120ul = 9.24ug (78.6% yield)
- V7: 61.0ng/ul x 120ul = 7.32ug (59.8% yield)
Remove Amplification Adapters
USER
- Split each set into 2 PCR tubes and add 5ul USER
Components | Volume |
SS-amplicon | 60 |
USER | 5 |
H2O | 20 |
Total | 85 |
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components | Volume |
10X DpnII Buffer | 10 |
100uM RE-DpnII guide oligo | 5 |
RE-DpnII guide oligo: V4: RE DpnII V4 V7: RE DpnII V7
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight
TBU Gel: Digestion Check
- Combine into 1 tube for each set
- 2ul Digested Sample + 2ul 2X Urea Loading Dye
- 5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
- 2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye)
File:2015-01-20 V4V7 Digest GelCheck.jpg
- V7 Undigested (middle lane) is extremely faint but can see band around 200bp
- Not sure why V4 lane is squiggly but can see the 3 bands
- V7 lane shows only 2 bands but both are below the undigested V7 band
Zymo Column Purification
- 2 columns for each set
- Eluted 30ul each column (60ul total)
- Nanodrop
- V4: 78.8ng/ul x 60ul = 4.7ug
- V7: 38.8ng/ul x 60ul = 2.3ug
PAGE Size Selection
- Run 2 gels for V4 and 1 gel for V7
- 200V for 30min
Components | 2X Volume |
V4 Probes | 120 |
TBE-Urea Buffer 2X | 120 |
Components | 1X Volume |
V7 Probes | 120 |
TBE-Urea Buffer 2X | 120 |
Components | Volume |
Low Mass Ladder | 3 |
TBE-Urea Buffer 2X | 15 |
H2O | 12 |
Total | 30 |