Editing
AlanFung:LabNotes/Probe/2009-5-1
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
==Purpose== *To improve my prob preping skills *Be extremely careful with contamination, clean every tools and equipments before use *Following kun's protocol exactly the way he did it on 04/16/09 ==Step 1: PCR reaction system setup== *Thaw Econo Taq on ice *Prepare 4 strips and 2x 2ml tubes on cooling rack *Add in Content in table to tubes accordingly {| border="1" align="center" | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''X32''' | align="center" style="background:#f0f0f0;"|'''2 mL Tube''' | align="center" style="background:#f0f0f0;"|'''2 mL Tube''' |- |RNAse free H2O||align="right"|49uL||align="right"|1568uL||align="right"|784uL||align="right"|784uL|| |- |- |50X SYBG I||align="right"|0.4uL||align="right"|12.8uL||align="right"|6.4uL||align="right"|6.4uL|| |- |- |AP1V6 AP2V6 Mix (50uM)||align="right"|0.2uL||align="right"|6.4uL||align="right"|3.2uL||align="right"|3.2uL|| |- |- |AP1V6 AP2V6 Mix (50uM)||align="right"|0.2uL||align="right"|6.4uL||align="right"|3.2uL||align="right"|3.2uL|| |- |- |Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|6.4uL(5uL)||align="right"|3.2uL||align="right"|3.2uL|| |- |- |Econo Taq||align="right"|50uL||align="right"|1600uL||align="right"|800uL||align="right"|800uL|| |- |} *Mix content following the order from top to bottom in 2x 2 mL tube vortex and spin down *Aliquot 100uL using P200 pipette to strip 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 *Skiped one cycle [[Image:20090501_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"|8000uL |- |3M NaOAC||align="right"|0.1X Sample Volume||align="right"|320uL |- |Glycoblue||align="right"|1/300X Sample Volume||align="right"|10.7uL |- |Sample||align="right"|Sample Volume||align="right"|3200uL |- |Total Volume |colspan="2" align="right"|11530.7uL |- |} *Pool all 32 wells PCR product into tray using multichannel pipette(P300) *Mix all content by repeat pipetting using 10mL Pipette *Aliquot to 2 x 15mL tubes *Store in -80C for more than 20 mins *Turn on Centrifuge fix temperature at 4C *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 350uL cold 75% Ethanol transfer pellet to 1.5mL tube *Addin another 350uL 75% ethanol wash the 15ml tube and transfer to 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 ==Step 3: Purify the reaction with Qiaquick columns== *Add 5X sample volume (500uL) of PB buffer into each tube *Vortex and spin down *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 air dry *Add in 50uL EB Buffer to each column let stand for 5min *Repeat centrifuge *Measure concentration with Nanodrop *'''Tube A 260/280 OD: 1.95 108.3ng/uL * 50uL''' *'''Tube B 260/280 OD: 1.96 122.5ng/uL * 50uL''' ==Step 4: Adapter Removal== Exonuclease treatment In a PCR tube *To the 50ul Qiaquick purified amplicons *add 10ul 10X Lambda Exo Buffer, 5ul 10x Lambda exo *Incubate at37C 30min>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 * Airdry the column/tube assemblies * Add in 50uL EB buffer to each column let stand for 5 min * Repeat centrifuge * Measure concentration with Nanodrop *'''Tube A 260/280 OD: 1.84 8.6ng/uL * 50uL *'''Tube B 260/280 OD: 1.93 15.9ng/uL * 50uL Incubation with USER enzyme: *Add 5 uL of USER (1K/ml)enzyme incubate for 30min on 37C. *'''Take out 2uL from each tube for quantification by running a TBU gel''' *PCR Tube A: 0.5uL 10bp ladder + 4.5uL ddh2o + 5uL 2X buffer *PCR Tube B: 2uL sample + 2uL ddh2o + 4uL 2X buffer *PCR Tube C 2uL sample + 2uL ddh2o + 4uL 2X buffer *Pre-run gel for 30min, flush wells with P1000 and P200 *Denature all 3 tubes at 75C for 30min *Transfer to cooling rack *Load samples and ladder to gel (Ladder_Tube A_Tube B) *Run gel for 30min at 200V *Stain gel with 4uL sybr gold *put on orbital shaker for 5 min *Take image [[Image:ZhangLab_2 2009-05-01 16hr 23min.jpg]] Annealing of DpnII oligo and incubation with DpnII. *add 10ul of 10X DpnII buffer and 5uL RE-DpnIIs guide oligo (100uM) *Perform 94C 2m > 0.1C/sec to 37C > add 5ul of DpnII restriction enzyme -> 37C 2 hours > 75C 20 min > 37 4 hours > 4C hold
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information