Editing
Noi/NOTES/2014-3-11
(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!
==== Procedures ==== : -Before starting, add 250ul of beta-mercaptoethanol to 50mL of Genomic Lysis Buffer to a final dilution of 0.5% (v/v). Vortex ZymoBead to resuspend : <u>Preparing lysate and binding DNA</u> :: - Add 200ul of '''Genomic Lysis Buffer''' to '''50ul samples''' in 1.5mL tube :: - Add 10ul '''ZymoBeads'''. Mix by inversion :: - Incubate at RT for 5 min :: - Centrifuge at 1,500xg for 1 min :: - Carefully remove the spnt without disturbing the bead pellet :: - Add 200ul of '''Genomic Lysis Buffer to the ZymoBeads :: Resuspend the beads by pipetting up & down :: Centrifuge at 1,500xg for 1 min :: - Carefully remove the spnt without disturbing the bead pellet : <u>Washing DNA</u> :: - Add '''200ul''' of '''DNA Pre-Wash Buffer''' to the ZymoBeads. :: - Resuspend the pellet, transfer to a new tube, centrifuge at 1,500xg for 1min :: - Carefully remove the spnt without disturbing the bead pellet :: Add '''500ul''' of '''g-DNA Wash Buffer''' to the ZymoBeads. :: - Resuspend the pellet and centrifuge at 1,500xg for 1 min. :: - Carefully remove the spnt without disturbing the bead pellet :: Re-centrifuge briefly and remove the residual wash buffer : <u>Eluting DNA</u> :: - Add '''35ul''' of '''Elution Buffer''' and resuspend the pellet by pipetting up & down. :: - Centrifuge at 10,000xg for 1 min :: - Collect the spnt to the 1.5mL tube tube. ---- '''<u>For bisulfite treatment</u>''' :: - After DNA binding step prior to pre-washing step, I resuspend the beads with 20ul of Elution Buffer :: - Add 130ul of Lightning Conversion Reagent and mix by pipetting 10X :: - Transfer solution to 200ul tube :: - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit :: - Elute with 17ul of Elution buffer --> volume after spin is ~16ul :: - Used 2ul for Qubit ssDNA assay :: - I will calculate total amount in 16ul --> normalize yield to 100ul input sample <u>Quant-iT ssDNA</u> :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Sample Conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Yield in 16ul (ng)''' |- | #1-P-Z||<1ng/mL|||||||||||| |- | #1-S-Z||1.3||ng/mL||2||100||130.00||pg/ul||1.82 |- | #1-BC-Z||12.6||ng/mL||2||100||1.26||ng/ul||17.64 |- | #2-P-Z||1.5||ng/mL||2||100||150.00||pg/ul||2.10 |- | #2-S-Z||3.1||ng/mL||2||100||310.00||pg/ul||4.34 |- | #2-BC-Z||38||ng/mL||2||100||3.80||ng/ul||53.20 |} <u>Normalized to 100ul sample input</u> :{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Volume used (ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ng)''' | align="center" style="background:#f0f0f0;"|'''Yields in 100ul (ng)''' |- | #1-P-Z||50||NA||NA |- | #1-S-Z||50||1.82||3.64 |- | #1-BC-Z||50||17.64||35.28 |- | #2-P-Z||50||2.10||4.20 |- | #2-S-Z||50||4.34||8.68 |- | #2-BC-Z||50||53.20||106.40 |} ---- : - For comparison of the four kits in DNA extraction efficiency, I am waiting for Richard's help for Bioanalyzer HS DNA measurement of 24 sample before continue to bisulfite conversion step. : - Richard helped me to do Bioanalyzer analysis. There were some samples that I suspected that the labeling may be wrong (between 2-P-Nu and 2-S-Nu).
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