User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/18: Difference between revisions
From OpenWetWare
Line 10: | Line 10: | ||
* Ben - assembly: HPK-CFP into homology arms | * Ben - assembly: HPK-CFP into homology arms | ||
* Cas-tone - modification #1 | * Cas-tone - modification #1 | ||
* Rene - PCR cloning trial 3 | |||
Line 161: | Line 162: | ||
| || # μL || # μL || | | || # μL || # μL || | ||
|} | |} | ||
---- | |||
'''Rene - PCR cloning trial 3''' | |||
* This time use Roche ligation buffer and NEB T4 ligase | |||
* Use the CloneJET PCR Cloning Kit - [https://tools.lifetechnologies.com/content/sfs/manuals/MAN0012707_CloneJET_PCR_Cloning_UG.pdf MAN0012707 CloneJET PCR Cloning (manual)] | |||
** pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products (Phusion PCR) | |||
** All common laboratory ''E. coli'' strains can be directly transformed with the ligation product | |||
** Re-circularized vector expresses toxic enzyme, no blue/white screening needed | |||
** Optimal insert : vector (0.05 pmol ends) ratio is 3:1; Calculation: '''length of insert DNA x 0.05 = ng insert to use''' | |||
* Ligations & transformations - CloneJET PCR Cloning Kit | |||
# Luc14 + CRISPR g034; size = 630 | |||
# Gal4EED/luc + CRISPR g034; size = 630 | |||
{| {{table}} cellspacing="3" | |||
|- | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
|- | |||
| 2x Roche lig buf || 10.0 | |||
|- | |||
| PCR product (>31.5 ng) || ## | |||
|- | |||
| pJET1.2/blunt vector || 1.0 | |||
|- | |||
| NEB T4 ligase || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 4.0 | |||
|- | |||
| || 20.0 μL | |||
|} | |||
* Incubate the ligation mixture at room temperature (22°C) for 5 min. | |||
* Transform 50 μL DH5α-turbo with 10 μL ligation reaction. Follow short protocol. | |||
RESULTS (5/15/15) | |||
* tba | |||
Revision as of 17:59, 18 May 2015
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
05/18/15
Ben - assembly: HPK-CFP into homology arms
Program: Phusion (block B)
Conclusion
Cas-tone Project
Assembly
--> 37°C/ ~30 min.
LabNet OptiMax Thermocycler: AnOlig RD
Dilute the product(s) 1:250
Rene - PCR cloning trial 3
|