Editing
AlanFung:LabNotes/CTCF/2011-8-26
(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!
==Continue on 8/30== ===PAGE Size Selection=== *Talked to Noi, she suggests that I can load ~500ng/well for my sample size is a a big range, I will load 2 wells per sample *Load a 5well TBE gel 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 Mix 1ul low mass ladder with 10ul dH2O, 9ul 6X loading dye Mix 6.5ul samples with 4.5ul dH2O, 9 ul 6X loading dye *Load the samples into the wells, run at 250V for 25 min. *Stain gel with 2.5uL SYBR Gold in ~100 mL of TBE in clean tray. *Place tray on an orbital shaker for ~5 min. ===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 (175-800 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly. [[File:ZhangLab_2 2011-08-30 15hr 04min.jpg|350px]] *Centrifuge at top speed > 13000 rcf 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 *Spin down all tubes at 13,000rcf 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. ===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. (35ul 3M NAoAC, 875ul 100% ETOH) *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 5 uL of water to each tube to resolve the DNA , leave at least 15 min. *Combine all 4 tubes into one ===Qubit Measurement=== **150cells:6ng/ul **300cells:10.7ng/ul
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