Editing
AlanFung:LabNotes/Probe/2009-5-12
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 *No SYBRgreen added *No column purification after ethanol precipitation *Dilute 24 wells 1 to 6 for column purification ==Step 1: PCR reaction system setup== *Thaw Econo Taq on ice *Prepare 6 strips and 4x 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;"|'''X48''' | align="center" style="background:#f0f0f0;"|'''2 mL Tube''' | align="center" style="background:#f0f0f0;"|'''2 mL Tube''' |- |RNAse free H2O||align="right"|50uL||align="right"|2400uL||align="right"|600uL||align="right"|600uL|| |- |- |AP1V6 AP2V6 Mix (100uM)||align="right"|0.2uL||align="right"|9.6uL||align="right"|2.4uL||align="right"|2.4uL|| |- |- |AP1V6 AP2V6 Mix (100uM)||align="right"|0.2uL||align="right"|9.6uL||align="right"|2.4uL||align="right"|2.4uL|| |- |- |Jan_09_CPG_D40809 (10uM)||align="right"|0.2uL||align="right"|9.6uL||align="right"|2.4uL||align="right"|2.4uL|| |- |- |Econo Taq||align="right"|54.17uL||align="right"|2600uL||align="right"|650uL||align="right"|650uL|| |- |} *Mix content following the order from top to bottom in 4x 2 mL tube vortex and spin down *Aliquot 104uL 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) X16 > 72C 3M > 15C Hold ==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"|12572uL |- |3M NaOAC||align="right"|0.1X Sample Volume||align="right"|502.88uL |- |Glycoblue||align="right"|1/300X Sample Volume||align="right"|16.76uL |- |Sample||align="right"|Sample Volume||align="right"|5028.8uL |- |Total Volume |colspan="2" align="right"|18120.44uL |- |} *Pool all 48 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 in 2 x 1.5mL tubes *take out 20uL from tube A perform qiaquick and nanodrop *OD 260/280:1.95 46ng/uL*50uL ==Step 4: Adapter Removal== Exonuclease treatment In a PCR tube *Combine the 50uL qiaqick purified amplicons with 80uL sample (tubeA) *To tube A add 4uL Lambda Exo, 13uL 10X Lambda Exo buffer *To tube B add 4uL Lambda Exo, 11uL 10X Lambda Exo buffer *Incubate at37C 30min>4C hold Saved as program Alan>EXOAR Purify the reaction with Quaquick columns *Aliquot both tube A and B in to 12 aliquots (~24uL per pcr tube) * Add 6X sample volume of PB buffer a 1.5mL 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 * For two tubes in A and two tubes in B add in 50uL EB buffer to each column let stand for 5 min *For the remaining tubes add in 30 uL EB buffer to each column let stand for 5 mins * Repeat centrifuge * Measure concentration with Nanodrop *A(50uL EB buffer) 260/280 OD:1.82 15.9ng/uL *A(50uL EB buffer) 260/280 OD:1.82 11.9ng/uL *A(30uL EB buffer) 260/280 OD:1.72 30.4ng/uL *B(50uL EB buffer) 260/280 OD:1.88 14.7ng/uL *B(30uL EB buffer) 260/280 OD:1.82 23.1.4ng/uL *Pool and aliquot 110uL sample to 4 pcr tube *Add in 2.5uL USER (1K/mL) enzyme into each tube incubate for 30m at 37C Annealing of DpnII oligo and incubation with DpnII. *Add 11uL of 10X DpnII buffer and 2.5uL RE-DpnIIs guide oligo (100uM) to each tube *Perform 94C 2m > 0.1C/sec to 37C > add 2.5ul of DpnII restriction enzyme to each tube -> 37C 2 hours > 75C 20 min > 37 4 hours > 4C hold ==Step 5: Page Denaturing Gel== *Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel *Load 2 gels into the gel running module *Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line *Pre-run the gel for 30 min at 200V *Dilute sample to no more than 40ng/uL *Dilute TBE-Urea sample buffer (2X) to 1X in each tube. For 2 gels; Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer *Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins *Flush the wells very well with P1000 & P200 pipette to remove all urea. '''NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP''' *Load the samples into the wells, run at 200V for 30 min. *Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray. *Place tray on an orbital shaker for ~5 min. ==Step 6: Purify the product from the gel== *Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel. *Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly. *Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. *Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. *Spin down all tubes at 13,000rpm for 2min *In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube. [[Image:ZhangLab_2 2009-05-14 11hr 58min.jpg]] ==Step 7: Precipitation== '''Turn on centrifuge let it cool down to 4C''' *Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol. *Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA. *Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette. *Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette. *Let dry in hood ~ 10 min. *Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min. Store at 4C if needed, spin down before proceeding ==Step 8: Quantification of DNA using denaturing gel and a quantitative ladder== Combine all tubes into a 1.5mL tube 1. Add 4uL of 5ng/uL 90-mer, 4ul of H2O and 8 uL of 2x TBE-Urea buffer 2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer 3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min. 4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. 5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min. Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL. 6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield. [[Image:ZhangLab_2 2009-05-14 19hr 03min.jpg]]
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