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 ==== :: - Add plasma and sample to 1.5mL tube (adjust to total 200ul). :: - Note: the volume of sample could be up to 1000ul ::{| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''1X PBS (ul)''' |- | #1-P-CS||50.00||150.00 |- | #1-S-CS||100.00||100.00 |- | #1-BC-CS||50.00||150.00 |- | #2-P-CS||50.00||150.00 |- | #2-S-CS||100.00||100.00 |- | #2-BC-CS||50.00||150.00 |} : - Mix 910ul of Lysis Buffer + 195 Proteinase K (140:30, 2 tubes) :: - Add '''170ul of Lysis Buffer and Proteinase K mix soulution (140ul of Lysis Buffer and 30ul of Proteinase K for 200ul sample) :: - Pipette up & down 5x to mix :: - Incubate at RT for 20min to lyse the sample. : <u>Binding DNA</u> : - Vortex the tube containing ChargeSwitch Magnetic beads : - Mix 50ul of ChargeSwitch Purification Buffer + 6ul of ChargeSwitch Magnetic beads for 13 rxn (650ul buffer + 78ul beads) :: - Add 56ul of ChargeSwitch Purification Buffer and ChargeSwitch Magnetic beads mix to the digested sample :: - Pipette up & down 5x to mix (use p1000 pipette, avoid generating bubbles) :: - Incubate at RT for 2 min to allow DNA bind to magnetic beads :: - Place the sample tube in the MagnaRack for 3 min or until the bead forming a tight pellet. (I may use magnetic rack for 1.5mL tube available in the lab) :: - Carefully remove the spnt without disturbing the bead pellet : <u>Washing DNA</u> :: - Remove sample tube from magnetic rack :: - Add 200ul of ChargeSwitch Wash Buffer to the tube and pipette up & down 5x gently & avoid bubbles :: - Place the sample tube in the MagnaRack for 2 min or until the bead forming a tight pellet :: - Carefully remove the spnt without disturbing the bead pellet :: - Repeat washing step : <u>Eluting DNA</u> :: - Remove sample tube from magnetic rack (there should be no spnt in the tube) :: - Add '''25ul''' of '''ChargeSwitch Elution Buffer''' to the tube and pipette up & down gently 10x to resuspend magnetic beads ::: # '''Do not use H2O for elution''' :: - Place the sample tube in the MagnaRack for 1 min or until the bead forming a tight pellet :: - Carefully transfer eluted DNA to 1.5mL tube ---- '''<u>For bisulfite treatment</u>''' :: - After DNA binding step prior to washing step, I resuspend the beads with 20ul of ChargeSwitch 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 3ul for Qubit ssDNA assay :: - Note: This kit was performed after Zymobeads. I saw that 2ul for assay was not enough to be detected for some samples, so I increase the sample volume from 2ul to 3ul. :: - 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-CS||<1ng/mL||||||||||||NA |- | #1-S-CS||<1ng/mL||||||||||||NA |- | #1-BC-CS||1.2||ng/mL||3||66.7||79.00||pg/ul||1.26 |- | #2-P-CS||<1ng/mL||||||||||||NA |- | #2-S-CS||3.1||ng/mL||3||66.7||210.00||pg/ul||3.36 |- | #2-BC-CS||64.6||ng/mL||3||66.7||4.31||ng/ul||68.96 |} <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-CS||50||||NA |- | #1-S-CS||100||||NA |- | #1-BC-CS||50||1.26||2.53 |- | #2-P-CS||50||||NA |- | #2-S-CS||100||3.36||3.36 |- | #2-BC-CS||50||68.96||137.92 |} <br>
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