Agi26k0gap Probe Production[edit]
Production PCR[edit]
- Make Master Mix for 51 well (7 strips)
- 0 gap first round amplicon -> V6 primers
Components
|
1x rxn (ul)
|
50x rxn (ul)
|
First round amplicon (10nM) |
0.2 |
10
|
2x Kapa SYBR MM |
50 |
2500
|
100uM pAP1V6U |
0.4 |
20
|
100uM AP2V6 |
0.4 |
20
|
H2O |
49 |
2450
|
Total |
100 |
5000
|
|
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold
File:2014-08-14 Agi26k0gapProductionPCR.JPG
EtOH Precipitation[edit]
- 6 15-ml tubes (with 8.5 wells of PCR product each) for 0gap
- 850ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 85ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for overnight
- Centrifuged at 3000rpm at 4C for 30min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
- Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
- Discarded supernatant and air-dried for 5 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification[edit]
- Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 40ul and combined all probes into one 1.5ml
- Measured concentration with Nanodrop:
- ~225ul of Agi26k0gap probes: 317.5 ng/ul => 71.4 ug
Lambda Exonuclease Digestion[edit]
- Divide into seven pcr tubes of 32ul with total amplicon of ~10ug each
- Seventh tube had 33ul of amplicon
Components
|
Volume
|
Amplicon |
32
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
48
|
Total |
100
|
- Incubated at 37C for 1hr
- Purified with Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 89.0ng/ul x 255ul = 22.7ug (63.6% yield)
Removal of Amplification Adapters[edit]
Components
|
Volume
|
ss-amplicon |
63.5
|
USER |
5
|
H2O |
11.5
|
total |
80
|
*~5ug ssDNA per tube
- Incubated at 37C for 2 hours
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
10
|
100uM RE-DpnII guide oligo |
5
|
RE-DpnII guide oligo:
0-gap: RE DpnII V6
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for 3 hr
- Added 100ul of the following
Components
|
Volume
|
10X DpnII Buffer |
10
|
USER |
5
|
H2O |
85
|
Total |
100
|
- Incubated at 37C for overnight (~15 hrs)
TBU Gel Check[edit]
- Combine the 4 tubes
- 2ul Sample + 2ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
- Digested sample
- Undigested sample: 10nM 1st round amplicon
File:2014-08-17 Agi26k0gapDigestCheck.jpg
- Brightest band below 200bp as expected
Column Purification[edit]
- Used Zymo ssDNA/RNA columns
- Total Eluted: 260 ul
- Nanodrop quant: 50.8 ng/ul
- Yield: 50.8 ng/ul x 260 ul = 13.2 ug
PAGE Size Selection[edit]
- On 8/26/14
- Run 4 gels of ~3.3ug ssDNA in each
- Corresponds to 65ul in each gel
Components
|
1X Volume
|
4X Volume
|
Enzyme digested probes |
65 |
260
|
TBE-Urea Buffer 2X |
65 |
260
|
Water |
0 |
0
|
Total |
130 |
520
|
Components
|
4X Volume
|
Low Mass Ladder |
4
|
TBE-Urea Buffer 2X |
20
|
H2O |
16
|
Total |
40
|
- Ran at 220V for 30min
- Cut out brightest band below 200bp
File:2014-08-26 Agi26k0gapSizeSelectionGel1.jpg
File:2014-08-26 Agi26k0gapSizeSelectionGel2.jpg
File:2014-08-26 Agi26k0gapSizeSelectionGel3.jpg
File:2014-08-26 Agi26k0gapSizeSelectionGel4.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 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 (~450ul per tube)
- Precipitated 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 overnight
- 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 (70ul total)
TBU Gel Quantification[edit]
- Diluted Agi26k0gap 10X
- 2ul Agi26k0gap + 8ul H2O + 10ul 2X TBE-Urea buffer
- Made DNA Low Mass Ladder with 0.5, 1, and 2 ul
Lane
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
Sample |
0.5ul DNA Ladder |
1ul DNA Ladder |
2ul Sample |
4ul Sample |
6ul Sample |
8ul Sample |
2ul DNA Ladder
|
File:2014-08-27 Agi26k0gapGelQuant.jpg
- Agi26k0gap Concentration: 82.4 ng/ul
- Total Mass: 82.4 ng/ul * 70ul = 5.77 ug
- Total Amount: 5.77 ug /(193nt*325 Da/nt) = 92 pmole (1.314 uM)