GP1 Padlock Probe Production[edit]
Overview[edit]
- Expansion PCR
- Production PCR
- Exonuclease digestion to generate SS-DNA
- Removal of amplification adapters
- Probe purification by PAGE size-selection
Expansion PCR[edit]
- Probe concentration from CustomArray 32.71ng/ul volume 80uL Amount 2.62ug
- Probe size 146bp
Convert ng/ul to nM
- Molar Mass of ssDNA probe: 146bp * (325Da/bp) = 146bp * (325 g/mol/bp) = 47450 g/mol
- (32.71ng/ul * 1*10^6 ul/L) / (47450g/mol) = 689.36nM
- I will use 100nM final concentration for the expansion PCR
GP1_V4 Expansion PCR[edit]
Components
|
1x rxn (ul)
|
Final Conc
|
GP-1 (689nM)
|
14.51
|
100nM
|
KAPA SYBR FAST Master Mix (2X)
|
50
|
1X
|
100uM AP1V41U
|
0.4
|
400nM
|
100uM AP2V4
|
0.4
|
400nM
|
H2O
|
34.69
|
|
Total
|
100
|
|
GP1_V6 Expansion PCR[edit]
Components
|
1x rxn (ul)
|
Final Conc
|
GP-1 (689nM)
|
14.51
|
100nM
|
KAPA SYBR FAST Master Mix (2X)
|
50
|
1X
|
100uM pAP1V61U
|
0.4
|
400nM
|
100uM AP2V6
|
0.4
|
400nM
|
H2O
|
34.69
|
|
Total
|
100
|
|
Realtime-PCR profile[edit]
File:12132013 GP1 V4.JPG
File:12132013 GP1 V6.JPG
- Left:GP1_V4 Right:GP1_V6
- 95C 30sec
- (95C 10sec → 55C 45sec → 70C 45sec) x 15 cycles
- 72C 3min
- Hold at 15C
Qiaquick Column Purification[edit]
Purify with one column per reaction[edit]
- In 1.5mL tube, add 6x volume of PB buffer to each tube with PCR product and mix (600ul to 100 ul of PCR product)
- Transfer 700ul of mixture to each qiaquick column
- spin at 14,000rpm for 1". Discard supernatant
- Add 750ul wash buffer to each column
- spin at 14,000rpm for 1". Discard supernatant
- repeat wash with wash buffer
- transfer column to 1.5mL lowbind tube
- Add 50ul 55C EB buffer to each column and incubate in 37C for 5"
- Elute DNA by spinning at 14,000 rpm for 1 min.
- Pool the purified amplicon into the same tube (total volume ~100ul).
- Measure DNA concentration by Nanadrop and calculate the total yield (dsDNA).
- Dilute purified 1st round amplicon to 10nM with TE buffer.
Nanodrop[edit]
GP1_V4
GP1_V6
- Left:GP1_V4 Right:GP1_V6
- GP1_V4:10ng/ul, 112.56nM
- GP1_V6:9.9ng/ul, 111.44nM
- Keep 1ul for Gel quantification
Dilute probes to 10nM[edit]
- GP1_V4: Add 0.85ul 112.56nM probes to 8.75ul EBuffer
- GP1_V6: Add 0.86ul 111.44nM probes to 8.74ul EBuffer
Production PCR (24 wells)[edit]
Mix the reaction mix in 50-mL tube
Mix well and aliquot 100ul of reaction mix into each well with multichannel pipettes.
GP1_V4 Production PCR[edit]
Components
|
1x rxn (ul)
|
24x rxn (ul)
|
10nM 1st round amplicon
|
0.4
|
9.6
|
KAPA SYBR FAST Master Mix (2X)
|
50
|
1200
|
100uM AP1V41U
|
0.4
|
9.6
|
100uM AP2V4
|
0.4
|
9.6
|
H2O
|
48.8
|
1171.2
|
Total
|
100
|
2400
|
GP1_V6 Production PCR[edit]
Components
|
1x rxn (ul)
|
24x rxn (ul)
|
1st round amplicon
|
0.4
|
9.6
|
KAPA SYBR FAST Master Mix (2X)
|
50
|
1200
|
100uM pAP1V61U
|
0.4
|
9.6
|
100uM AP2V6
|
0.4
|
9.6
|
H2O
|
48.8
|
1171.2
|
Total
|
100
|
2400
|
Realtime-PCR profile[edit]
File:12132013 GP1 V4 production.JPG
File:12132013 GP1 V6 production.JPG
- Left:GP1_V4 Right:GP1_V6
- 95C 30sec
- (95C 10sec → 55C 20sec → 72C 30sec) x 15 cycles
- 72C 2min
- Hold at 15C
EtOH precipitation[edit]
- Transfer the amplified amplicon from 96-well plate into 50-mL disposable basins (Total volume ~9.6mL)
- Aliquot 1.2 mL of amplified amplicon into 15-mL tube (4 tubes)
- In each tube, add 4 ul of Glycoblue, 120 ul of 3M NaOAc, and 3 mL of 100 % EtOH (2.5 volume of 100% EtOH, 1/300 volume of Glycoblue, 0.1 volume of 3M NaOAc pH 5.2 or 5.5)
- Mix well and leave at -80C for at least 30 min.
- Precipitate by centrifugation at ~10,000xg for 30 min, at 4 C
- Discard spnt and add 800ul of cold 75% EtOH to each tube.
- Use 1 mL clean pipette tip transfer DNA pellet to 1.5 mL tube.
- Rinse the pellet and spin the tube at 14,000 rpm for 5 min.
- Discard spnt and air-dry the pellet for 3-5 min in the hood (PCR work station).
- Resuspend DNA with 150 ul ddH2O mix well by pipetting up and down.
Qiaquick Re-purification[edit]
- Add 6x volume of PB buffer to each tube (900ul to 150 ul of DNA)
- Transfer 700 ul of the mixture to each QIAquick column (6 columns total, 3 for each set)
- Spin at 14,000 rpm for 1 min. Discard the spnt.
- Add 750 ul of PE buffer to each tube.
- Spin at 14,000 rpm for 1 min. Discard spnt (repeat washing step with buffer PE)
- Spin the empty column at 14,000 rpm for 1 min.
- Transfer the column to a fresh 1.5 mL tube.
- Add 50 ul of 55C EB buffer to each tube
- Elute the DNA by spinning at 14,000 rpm for 1 min.
- Pool the purified amplicon into the same tube (total volume ~100 ul for each set).
- Measure DNA concentration by Nanadrop and calculate the total yield (ds-DNA).
Nanodrop[edit]
File:12142013 GP1 V4 ND.JPG
File:12142013 GP1 V6 ND.JPG
- Left: GP1_V4 Right GP1_V6
- GP1_V4: 18.1ng/ul *150ul = 2715 ng
- GP1_V6: 20.6ng/ul *150ul = 3090 ng
Exonuclease digestion to generate SS-DNA[edit]
Components
|
1x rxn (ul)
|
2x rxn (ul)
|
GP1_V4 Purified amplicon (~ 15ug)
|
75
|
150
|
10X Lambda Exonuclease Buffer
|
15
|
30
|
Lambda Exonuclease
|
10
|
20
|
H2O
|
50
|
100
|
Total
|
150
|
300
|
|
|
|
Components
|
1x rxn (ul)
|
2x rxn (ul)
|
GP1_V6 Purified amplicon (~ 15ug)
|
75
|
150
|
10X Lambda Exonuclease Buffer
|
15
|
30
|
Lambda Exonuclease
|
10
|
20
|
H2O
|
50
|
100
|
Total
|
150
|
300
|
- Incubate at 37 C for 1 hr on thermocycler
- Purify with each set in 3x QIAquick columns as described above (may use 7X volume of buffer PB for ssDNA
- Elute each column with 40 ul EB buffer (Total volume ~ 240 ul)
Nanodrop ssDNA[edit]
File:12142013 GP1 V4 ND SS.JPG
File:12142013 GP1 V6 ND SS.JPG
- Left: GP1_V4_SS Right: GP1_V6_SS
- GP1_V4_SS: 2.3 ng/ul *120ul = 276ng
- GP1_V6_SS: 3.3 ng/ul *120ul = 396ng
- Yield is too low, may be too much lambda exo for the reaction and lead to over digestion?
- Store probes in -20C for storage and troubleshoot
Removal of amplification adapters[edit]
- SETUP RXN
- Incubate at 37 C for 3 hr on thermocycler.
- Add 15 ul of the following components to the USER digested DNA tubes
- On thermocycler, incubated at 94 C for 2 min, cool down temperature to 37C for 3 min
- Keep the tube on thermocycler.
- Add 5 ul of DpnII and mix well by pipetting up and down
- Incubate at 37 C for 2 hr.
Qiaquick Purification[edit]
- Purify USER/DpnII digested amplicon with 4-6 QIAquick columns as described previously (use 7X volume of buffer PB).
Nanodrop ssDNA[edit]
- Measure DNA concentration (ssDNA) by Nanodrop and calculate total yield.
Probe purification by PAGE size-selection[edit]
EtOH Precipitation[edit]
Qubit ssDNA[edit]