Editing
Dinh:Probes Prep
(section)
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!
= Padlock probes preparation = == Production PCR == * I usually prepare 20x 100 uL reactions in a 2.0 mL tube. (For 96 wells plate, prepare 100 reactions & aliquot 104 uL to each). * qPCR Reaction: KAPA Master Mix(2x) : 1000 uL H2O : 980 uL AP1V4/6(100uM) : 8 uL AP2V4/6(100uM) : 8 uL Template (10nM) : 4 uL * Program: 95C for 30s -> [95C for 10 s -> 55C for 20s -> 72C for 30s] x 13-15 cycles -> 72C for 2 minutes == Ethanol precipitation == * Pool 20 reactions into 2x 15 mL tube * Add 2.5x 100% ethanol, 0.1x 3M sodium acetate, and 8 uL of glycoblue * Store in -80C for at least 1 hour. * Spin at >4000 rpm for 40 minutes at 4C * Remove supernatant & add 1 mL 80% ice-cold ethanol * Spin at >4000 rpm for 15 minutes at 4C * Remove supernatant & air dry for 10 minutes * Add 150 uL of water to each tube & pool together. * Purify with 3x Qiaquick PCR cleanup columns. (Optional: dry residual ethanol by warming columns in thermomixer for 5 minutes at 50C). Elute each with 30 uL of pre-warmed EB. * Nanodrop to quantify yield. == Removal of amplification adapters == * NOTE: Make sure Lambda exo reaction is complete (check with TBE gel, should see 1 ssDNA band, not two) & use fresh DpnII buffer & use DpnII at 50 units/uL to get the best digestion efficiency. * Lambda Exonuclease reaction: Lambda exo buffer (10x) : 15 uL Lambda exo (5 units/uL) : 10 uL dsDNA probes (up to ~20 ug) : 125 uL * Incubate reaction at 37C for 1 hour. * Purify with 2x ssDNA/RNA cleanup columns. Elute each with 30 uL of EB. * Nanodrop to quantify yield. * USER digestion reaction: USER (1 unit/uL, NEB) : 8 uL DpnII buffer (10x) : 10 uL ssDNA probes (up to 5 ug) : 82 uL (100 ng/uL is 2.02 pmol/uL, 150 nt oligos) * 1 unit of USER digests 10 pmol of uracils at 37C for 15 minutes. * Incubate reaction at 37C for at least 1 hour. * Prepare DpnII oligo mix: RE-DpnII_2N V4/6 guide oligo : 12 uL DpnII buffer (10x) : 2 uL (Keep DpnII buffer on ice or frozen when storing, do not use very old DpnII buffer) H2O : 5 uL * Add 19 uL of DpnII oligo mix to each USER reaction mix. * Program: 94C for 5 minutes -> 60C for 20 min, ramp -0.1C/s to 37C -> 37C for 3 minutes * Add 1 uL of DpnII (50 units/uL) while tubes are at 37C * Incubate at 37C for 2 hours. * Add more DpnII (important if >= 5 ug of ssDNA was used): DpnII (NEB, 50 units/uL) : 1 uL DpnII buffer (10x) : 1 uL H2O : 8 uL * Add 10 uL to each reaction. * Incubate at 37C for one hour. During the second USER digestion, ~5 uL of the digestion reaction can be checked for digestion completion. * Purify with 1x ssDNA/RNA cleanup column for each reaction tube. (Optional: dry residual ethanol by warming columns in thermomixer for 5 minutes at 50C). Elute with 30 uL of pre-warmed EB. == PAGE size selection == * UV all gel running buffer and chamber. * UV shearing apparatus. ** Prepare 3x (2.0 mL tube + 0.5 mL tube) per gel. Cut off the caps of 2.0 mL tube to enable centrifuge at 15,000 rpm. * Puncture 0.5 mL gel with 20 gauge needle. * Run 6% gel for 40 minutes at 200 V. (Run lower volt to get better band resolution). * Add 1.5 mL TE buffer to sheared gel. * Rotate overnight at room temperature. * 50C for 15 minutes at 900 rpm on thermomixer. (Or 37C for 1 hour at 900 rpm). Yield with 37C/1 hour seems decent, not sure which is better. * Quickly spin-down tubes & filter through 1-2 Nanosep columns. Keep tube warm. * Pool flow-through in 1x 15 mL tube. * Add 2.5x 100% ethanol, 0.1x 3M sodium acetate, and 8 uL of glycoblue * Store in -80C for at least 1 hour. * Spin at >4000 rpm for 40 minutes at 4C * Remove supernatant & add 1 mL 80% ice-cold ethanol. Transfer to 1x 1.5 mL tube. * Spin at >10,000 rpm for 15 minutes at 4C * Remove supernatant & vacumm dry for 10-30 minutes * Add 30 uL of pre-warmed nuclease-free water. * QUBIT ssDNA to quantify yield. == Agarose gel size selection == * Prepared >4% agarose gel (Apex multipurpose agarose). 1 g in 25 mL. UV for 15 minutes. * Put on heater/mixer at 200 degC let sit for about 5-10 minutes. Turn off the heat. * Fill a 2 L beaker partly with water and put the flast with melted agarose inside. Continue stirring for 10 minutes to cool the agarose solution. * Added 2uL of Sybr Gold per 25mL of agarose solution when the agarose solution have cooled enough to hold for 15 seconds. * Cast a gel of about 7 mm thickness with 3-5 mm wells. * Load at least 700 ng into each well. Purification will not work with less than 700 ng minimum input. * Ran at 135V for 45 minutes, the probes were in the bottom quarter of the gel by the end of electrophesis run. * Gel image: * After sample finished running. Weight a 50 mL tube. * Cut out 2 wells per tube and re-weigh to calculate 6X volume of QX1 buffer to add to each tube. * Add 10 uL of QIAX suspension solution to each tube. * Incubate at 60 degC for 6 minutes. Vortex every 2 minutes. * Label 2 mL tubes for spinning down the solution. Remove supernatant after each spin. * Wash with 500 uL QX buffer once. * Wash with 500 uL PE buffer three times. * Dry the pellet in the vacuum for 2 minutes. * Resuspend the pellet with water.
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