Editing
Brandon:LabNotes/Project1/2014-6-18
(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!
==Dialysis of last fractions, and Tn5 activity testing== *Dialysis was performed on the last tn5 fractions from the chromatography column. This includes the fractions obtained from letting the column sit overnight in 3-4 mL cleavage buffer after collection of the first fractions. **8.44 uM was isolated from the last fractions **7.186 uM was isolated from the column left overnight *will test the fractions for enzyme activity as it is hoped that there will still be tn5 left over, otherwise it will have to be isolated again. ===Before starting protocols=== 1. Check if have enough reagents etc for the protocol *2. Samples *transpoase was either directly annealed to transposon, diluted in storage buffer then annealed to transposon, diluted 2X more after being diluted in storage buffer, or 4X more after being diluted in storage buffer. samples (all use buffer D6) 1. T7tspn-top2, E1 direct anneal (4.22 uM), 6 ng DNA 2. T7tspn-top2, E1 strg-anneal (1.77 uM), 6 ng DNA 3. T7tspn-top2, E1 2X dil. strg-anneal (.88 uM), 6 ng DNA 4. T7tspn-top2, E1 4X dil. strg-anneal (.44 uM), 6 ng DNA 5. T7tspn-top2, E2 direct anneal (3.6 uM), 6 ng DNA 6. T7tspn-top2, nxta direct anneal, 6 ng DNA 7. T7tspn-top2, nxta strg-anneal, 6 ng DNA 8. T7tspn-top2, nxta 2X dil. strg-anneal, 6 ng DNA 9. T7tspn-top2, nxta 4X dil. strg-anneal, 6 ng DNA 10. pure DNA only, 6 ng DNA 11. NTC ===Protocol=== *If need to make more transposome, do first 2 steps. If not goto step 3. '''Generation of sc1-T7iBR-IdxXX transposomes''' 1. annealing of ME sequence to T7 transposon sequence **a. Make 100 uM stock solution of sc1-T7iBR-IdxXX and sc1-T7tspn-NoP-bot. **b. Incubate 2.5 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 uL. **c. cool to RT at 0.1 C/s 2. transposome complex generation, run controls!!! *'''add the below components into one tube and incubate for 30 minutes at 37C (DID THIS TIME)''' 1. Add 0.5 uL of 100% sterile glycerol to tube 2. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well 4. Add 1.0 uL of '''DILUTED''' Ez-TN5 transposase to well. *store at -20, is good for a year 3. Prepare samples, lyse cells with lysis buffer 4. transposition reaction Add all components and incubate at 55C for 6 minutes 1.0 uL 5X Custom Tagmentation buffer 2.0 uL lysed cells/pure genomic DNA 1.0 uL (or .6 uL undiluted) prepared T7 transposomes (MAKE SURE TO ADD LAST) (5X diluted) 1.0 uL Nuclease free water ___________ 5.0 uL total solution 5. Protease digestion of proteins/transposase To each tube, add: 1 uL Proteinase K Incubate: 50C 10 minutes, '''Did not do 70C inactivation of protease''' ===Results=== *TBE gel *did not work. [[File:ZhangLab 2 2014-06-18 19hr 30min-labeled.jpg|600px]] ===conclusions=== *isolatedtn5 did not work while nextera tn5 did. Thus will have to reisolate *other possibility is T7tspn-top2 could be getting old and not working, thus will try reannealing to nextera tn5 as a control and testing again with purified tn5.
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