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!
==Cloning for Sequencing== ===Blunting with NEB end repair kit=== *Take 8.5ul of PCR product {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''150''' | align="center" style="background:#f0f0f0;"|'''300''' |- | Fragmented DNA||8.5||8.5 |- | 10X End repair reaction buffer||1||1 |- | End reapir enzyme||0.5||0.5 |- | H2O||0||0 |- | Total Volume||10||10 |} *Incubate at 20C for 30min *Minelute with 10ul EB ===Qubit=== **150cells:2.84ng/ul **300cells:5.12ng/ul ===Ligating into pCR blunt vector=== **Calculate the amount of PCR product needed for ligation *Talked to Rui and she used 5ng vector, I will try the same protocol as I got too many colonies from the previous cloning, however I will maintain insert to vector ratio at 10:1 **x ng insert = (10*500*5/3500) **X=7.14ng needed *Make 5ng/ul vector (Mix 2ul vector with 8ul water) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''150 cells''' | align="center" style="background:#f0f0f0;"|'''300 cells''' |- | pCR-Blunt (5ng/ul)||1||1 |- | Blunt PCR Product||2.51||1.39 |- | 10X ligation buffer (with ATP)||1||1 |- | Sterile water||4.49||5.61 |- | T4 DNA Ligase (4U/ul)||1||1 |- | Total Volume||10.00||10.00 |} *Incubate at 16C for 1 hr ===Heat-shock transfection=== *Equilibrate water bath to 42C. *Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min. *Thaw the TOP10 E.coli competent cell vial on ice and split 25ul to a new 1.5ml tube *Transfer '''1uL''' ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. ('''Mixing by repeat pipetting is prohibited in the protocl'''). *Set up conpetent cell vials in the foam-board holder(for water bath). Incubate the vials on ice for 30 mins. *Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min. *Add 125ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall). *Shake the vials horizontally at 37C for 1 hour using vortex in the incubator at the lowest speed. *During the 1hr incubation. *After 1hr shacking mixing, plate '''50uL''' from each transformation vial on agar plates (w/ Kanamycine and xgal) *Incubate plates at 37C for about 20hr.
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