Matt:LabNotes/2015-1-15: Difference between revisions
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*V4 | |||
**2ul sample lane was loaded poorly so was not used in concentration calculations | |||
[[File:2015-01-24_V4_GelQuant.jpg|650px]] | |||
*V7 | |||
**Only use ladder bands 100bp, 200bp, and 400bp | |||
[[File:2015-01-21_V7_GelQuant.jpg|650px]] | [[File:2015-01-21_V7_GelQuant.jpg|650px]] | ||
*V4: 65.3 ng/ul => 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul) | |||
*V7: 39.2 ng/ul => 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul) | *V7: 39.2 ng/ul => 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul) | ||
[[Media:2015-01-21_V7_GelQuant.xlsx | V7 Concentration calculation]] | [[Media:2015-01-21_V7_GelQuant.xlsx | V7 Concentration calculation]] | ||
[[Media:2015-01-24_V4_GelQuant.xlsx | V4 Concentration calculation]] |
Latest revision as of 22:34, 24 January 2015
CA12k_Nov2014 V4 and V7 Probe Production[edit]
- Repeat Matt:LabNotes/2015-1-5
Production PCR[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
USER[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- Run 2 gels for V4 and 1 gel for V7
- 200V for 30min
Components | 2X Volume |
V4 Probes | 60 |
TBE-Urea Buffer 2X | 60 |
Components | 1X Volume |
V7 Probes | 60 |
TBE-Urea Buffer 2X | 60 |
Components | Volume |
Low Mass Ladder | 3 |
TBE-Urea Buffer 2X | 15 |
H2O | 12 |
Total | 30 |
- V4 Gel 1
File:2015-01-20 SizeSelect V4 Gel1.jpg
- V4 Gel 2
File:2015-01-20 SizeSelect V4 Gel2.jpg
- V7 Gel 1
File:2015-01-20 SizeSelect V7 Gel1.jpg
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 2.0 mL tube below with pipette tip.
- Added 900 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 6 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 (~450ul per tube except for 1 V4 tube which spilled)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 6 tubes at -80C for overnight
- Spun 6 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 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 recombine (40ul for V4 and 20ul for V7)
TBU Gel Quantification[edit]
- Dilute 2ul to 10ul with H2O
- Add 10ul 2X TBE-Urea Buffer
- Load wells with 2, 4, 6, and 8 ul
- Load Low Mass DNA ladders in 0.5, 1, and 2 ul
- Ran at 200 V for 35min
' | 0.5 ladder | 1 ladder | 2 ul | 4 ul | 6 ul | 8 ul | 2 ladder |
DNA volume | 0.5 | 1 | 0.1 | 0.2 | 0.3 | 0.4 | 2 |
H2O | 1.5 | 1 | 0.9 | 1.8 | 2.7 | 3.6 | 0 |
2X Buffer | 2 | 2 | 1 | 2 | 3 | 4 | 2 |
Total | 4 | 4 | 2 | 4 | 6 | 8 | 4 |
- V4
- 2ul sample lane was loaded poorly so was not used in concentration calculations
File:2015-01-24 V4 GelQuant.jpg
- V7
- Only use ladder bands 100bp, 200bp, and 400bp
File:2015-01-21 V7 GelQuant.jpg
- V4: 65.3 ng/ul => 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul)
- V7: 39.2 ng/ul => 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul)