AlanFung:LabNotes/Probe/2009-4-23: Difference between revisions
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*'''Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube | *'''Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube''' | ||
*Add in (1/10 of total volume) 6uL 10X Exo buffer | *'''Add in (1/10 of total volume) 6uL 10X Exo buffer''' | ||
*Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR''' | *'''Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR''' | ||
Purify the reaction with Quaquick columns | Purify the reaction with Quaquick columns | ||
Line 23: | Line 23: | ||
* Repeat centrifuge | * Repeat centrifuge | ||
* Measure concentration with Nanodrop | * Measure concentration with Nanodrop | ||
*'''Tube A (04/22/09-1) 260/280 1.9 81.4ng/uL''' | |||
*Add | *'''Tube B (04/22/09-1) 260/280 1.81 74.4ng/uL''' | ||
*'''Tube C (04/22/09-2) 260/280 1.85 73.1ng/uL''' | |||
*'''Tube D (04/22/09-2) 260/280 1.88 69.63ng/uL''' | |||
*'''Tube A (04/22/09-1) 260/280 1.84 7.9ng/uL''' | |||
*'''Tube B (04/22/09-1) 260/280 1.78 8.5ng/uL''' | |||
*'''Tube C (04/22/09-2) 260/280 1.66 8ng/uL''' | |||
*'''Tube D (04/22/09-2) 260/280 1.76 6.4ng/uL''' | |||
* Concentration too low, probably due to over digest by Lambda Exo , next time run 24 wells to one tube | |||
*Combine Tube A(4/21) B(4/21) D(4/22-2) '''[[TUBE A]]''' | |||
*Combine A(4/22-1) B((4/22-1) C(4/22-2) by qiaquick column '''[[TUBE B]]''' | |||
* pool 3 tubes into one | |||
*add in 750uL PB buffer mix by repeat pipetting | |||
*load 450 mix to column | |||
*Spin down @ 14,000 rpm 1m | |||
*Discard flow through | |||
*Load the remaining 450uL into column | |||
*spin down @14,000 rpm 1m | |||
*Add in 750ul PE wash buffer | |||
*Spin down @ 14,000rpm 1m | |||
*Discard flow through | |||
*Repeat centrifuge | |||
*Place into new collection tube | |||
*Leave air dry for 5 m | |||
*add in 50uL DDH2O | |||
*Sit for 5m | |||
* spin down at 14,000rpm for 1m | |||
*Measure with Nanodrop | |||
*Decided to restart experiment, with alice's primer and new tube of lambda exo | |||
==Step 1: PCR reaction system setup 48 wells== | |||
*Thaw Econo Taq on ice | |||
*Prepare strips and tubes on cooling rack | |||
*Add in Content in table to tubes accordinly | |||
{| border="1" align="center" | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''X24''' | |||
| align="center" style="background:#f0f0f0;"|'''X48''' | |||
| align="center" style="background:#f0f0f0;"|'''X72''' | |||
| align="center" style="background:#f0f0f0;"|'''X96''' | |||
| align="center" style="background:#f0f0f0;"|'''1.5 mL Tube''' | |||
|- | |||
|Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|4.8uL||align="right"|9.6uL||align="right"|14.4uL||align="right"|19.2uL||align="right"|2.4uL | |||
|- | |||
|Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|650uL | |||
|- | |||
|AP1V6 AP2V6 Mix (50uM)||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL | |||
|- | |||
|RNAse free H2O||align="right"|50uL||align="right"|1200uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL||align="right"|600uL | |||
|- | |||
|50X SYBG I||align="right"|0.4uL||align="right"|9.6uL||align="right"|19.2uL||align="right"|28.8uL||align="right"|38.4uL||align="right"|4.8uL | |||
|- | |||
|Total Volume|| align="right"| 101uL||align="right"|2424uL||align="right"|4848uL||align="right"|7272uL||align="right"|9696uL||align="right"|1212uL | |||
|} | |||
*Mix content in 1.5 mL tube vortex and spin down in the order of water>SYBR Green>Primer >template>econotaq | |||
*Aliquot 105uL using P200 pipette to strip tubes on cooling rack, leave unused tubes on cooling rack | |||
*Seal strips with strip caps | |||
*Place into Real Time PCR | |||
*Perform | |||
94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X16 > 72C 3M > 15C Hold | |||
[[Image:20090423_CPGD40809.jpg]] | |||
==Step 2: Probe purification with ethanol precipitation== | |||
*Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray | |||
{| border="1" align="center" | |||
| align="center" style="background:#f0f0f0;"|'''Content''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume Needed''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
|100% Ethanol||align="right"|2.5X Sample Volume||align="right"|6000uL | |||
|- | |||
|3M NaOAC||align="right"|0.1X Sample Volume||align="right"|240uL | |||
|- | |||
|Glycoblue||align="right"|1/300X Sample Volume||align="right"|8uL | |||
|- | |||
|Sample||align="right"|Sample Volume||align="right"|2400uL | |||
|- | |||
|Total Volume | |||
|colspan="2" align="right"|8648uL | |||
|- | |||
|} | |||
*Mix all content by repeat pipetting using multichannel pipette | |||
*Pool all 24 wells PCR product into tray using multichannel pipette(P300) | |||
*Mix all content by repeat pipetting using multichannel pipette | |||
*Aliquot about 3750uL to 2 x 15mL tube | |||
*Store in -80C for more than 20 mins or forever | |||
*Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge | |||
*Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C | |||
*Remove all liquid '''CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE''' | |||
*Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube | |||
*Centrifuge at 10,000rpm for 5min at 4C | |||
*Remove all liquid, air dry sample for 5min or until samples dried out | |||
*Resuspend the DNA with 100uL RNAse free H2O | |||
*Break pellet by repeat pipetting | |||
*store in 4C overnight | |||
==Step 3: Purify the reaction with Qiaquick columns== | |||
*Add 5X sample volume of PB buffer into each tube (500uL) | |||
*Mix by repeat pipetting | |||
*Load mixture onto column | |||
*Centrifuge at 14,000rpm for 1min | |||
*Discard flow through and add 750uL PE buffer to each column | |||
*Repeat Centrifuge | |||
*Discard flow through | |||
*Repeat centrifuge | |||
*Transfer columns to clean 1.5mL tubes | |||
*Place the column/tube assemblies on bench top wait for 2min | |||
*Add in 50uL ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min | |||
*Repeat centrifuge | |||
*Measure concentration with Nanodrop |
Latest revision as of 15:58, 24 April 2009
Step 4: Adapter Removal[edit]
Exonuclease treatment
Done on 4/22/09
- Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube
- Add in (1/10 of total volume) 6uL 10X Exo buffer
- Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR
Purify the reaction with Quaquick columns
- Add 5X sample volume of PB buffer into each tube, mix well
- Load mixture onto column
- Centrifuge at 14,000rpm for 1min
- Discard flow through and add 750uL PE buffer to each column
- Repeat Centrifuge
- Discard flow through
- Repeat centrifuge
- Transfer columns to clean 1.5mL tubes
- Place the column/tube assemblies on bench top wait for 2min
- Add in 50uL ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
- Repeat centrifuge
- Measure concentration with Nanodrop
- Tube A (04/22/09-1) 260/280 1.9 81.4ng/uL
- Tube B (04/22/09-1) 260/280 1.81 74.4ng/uL
- Tube C (04/22/09-2) 260/280 1.85 73.1ng/uL
- Tube D (04/22/09-2) 260/280 1.88 69.63ng/uL
- Tube A (04/22/09-1) 260/280 1.84 7.9ng/uL
- Tube B (04/22/09-1) 260/280 1.78 8.5ng/uL
- Tube C (04/22/09-2) 260/280 1.66 8ng/uL
- Tube D (04/22/09-2) 260/280 1.76 6.4ng/uL
- Concentration too low, probably due to over digest by Lambda Exo , next time run 24 wells to one tube
- Combine Tube A(4/21) B(4/21) D(4/22-2) TUBE A
- Combine A(4/22-1) B((4/22-1) C(4/22-2) by qiaquick column TUBE B
- pool 3 tubes into one
- add in 750uL PB buffer mix by repeat pipetting
- load 450 mix to column
- Spin down @ 14,000 rpm 1m
- Discard flow through
- Load the remaining 450uL into column
- spin down @14,000 rpm 1m
- Add in 750ul PE wash buffer
- Spin down @ 14,000rpm 1m
- Discard flow through
- Repeat centrifuge
- Place into new collection tube
- Leave air dry for 5 m
- add in 50uL DDH2O
- Sit for 5m
- spin down at 14,000rpm for 1m
- Measure with Nanodrop
- Decided to restart experiment, with alice's primer and new tube of lambda exo
Step 1: PCR reaction system setup 48 wells[edit]
- Thaw Econo Taq on ice
- Prepare strips and tubes on cooling rack
- Add in Content in table to tubes accordinly
Content | Volume | X24 | X48 | X72 | X96 | 1.5 mL Tube |
Jan_09_CPG_D40809 (10uM) | 0.2uL | 4.8uL | 9.6uL | 14.4uL | 19.2uL | 2.4uL |
Econo Taq | 50uL | 1250uL | 2400uL | 3600uL | 4800uL | 650uL |
AP1V6 AP2V6 Mix (50uM) | 0.4uL | 9.6uL | 19.2uL | 28.8uL | 38.4uL | 4.8uL |
RNAse free H2O | 50uL | 1200uL | 2400uL | 3600uL | 4800uL | 600uL |
50X SYBG I | 0.4uL | 9.6uL | 19.2uL | 28.8uL | 38.4uL | 4.8uL |
Total Volume | 101uL | 2424uL | 4848uL | 7272uL | 9696uL | 1212uL |
- Mix content in 1.5 mL tube vortex and spin down in the order of water>SYBR Green>Primer >template>econotaq
- Aliquot 105uL using P200 pipette to strip tubes on cooling rack, leave unused tubes on cooling rack
- Seal strips with strip caps
- Place into Real Time PCR
- Perform
94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X16 > 72C 3M > 15C Hold File:20090423 CPGD40809.jpg
Step 2: Probe purification with ethanol precipitation[edit]
- Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content | Volume Needed | Volume |
100% Ethanol | 2.5X Sample Volume | 6000uL |
3M NaOAC | 0.1X Sample Volume | 240uL |
Glycoblue | 1/300X Sample Volume | 8uL |
Sample | Sample Volume | 2400uL |
Total Volume | 8648uL |
- Mix all content by repeat pipetting using multichannel pipette
- Pool all 24 wells PCR product into tray using multichannel pipette(P300)
- Mix all content by repeat pipetting using multichannel pipette
- Aliquot about 3750uL to 2 x 15mL tube
- Store in -80C for more than 20 mins or forever
- Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
- Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C
- Remove all liquid CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE
- Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube
- Centrifuge at 10,000rpm for 5min at 4C
- Remove all liquid, air dry sample for 5min or until samples dried out
- Resuspend the DNA with 100uL RNAse free H2O
- Break pellet by repeat pipetting
- store in 4C overnight
Step 3: Purify the reaction with Qiaquick columns[edit]
- Add 5X sample volume of PB buffer into each tube (500uL)
- Mix by repeat pipetting
- Load mixture onto column
- Centrifuge at 14,000rpm for 1min
- Discard flow through and add 750uL PE buffer to each column
- Repeat Centrifuge
- Discard flow through
- Repeat centrifuge
- Transfer columns to clean 1.5mL tubes
- Place the column/tube assemblies on bench top wait for 2min
- Add in 50uL ddH2O to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min
- Repeat centrifuge
- Measure concentration with Nanodrop