User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/28

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Entry title)
m (Hypothesis 2: Gene L is necessary for phage propagation.)
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
===Hypothesis 2: Gene L is necessary for phage propagation.===
 
-
 
-
* Last time, I verified the final WP-PCR protocol.
 
-
* Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
 
-
* For there to be 0.5 μg of DNA to digest in 10 μL, which is enough to see on a gel, I'll need 50 ng/μL ~ 15 nM final concentration DNA.
 
-
* 50 μL WP-PCR reaction w/o primers:
 
-
** 37 μL H<sub>2</sub>O
 
-
** 7.5 μL 100 nM ΦX174 template (~15 nM final)
 
-
** 5 μL 10X reaction buffer
 
-
** 0.5 μL 25 mM dNTPs mix
 
-
** 1 μL PfuUltra I DNA polymerase
 
-
 
-
 
-
-----------------
 
-
 
-
* Next on the list:
 
-
*# DpnI digestion (w/ and w/o PCR purification)
 
-
*# Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
 
-
* Final experiment is planned to be WP-PCR of 0.1 nM ΦX174 at optimized conditions (primer concentration, T<sub>a</sub>, elongation time, N), + PFU ligase
 
-
** experimental 1 = +template, +primer 4, +DNAP
 
-
** experimental 2 = +template. +primer 4 T3585A, +DNAP
 
-
** control 1: -template, +primer 4, +DNAP
 
-
** control 2: -template, +primer 4 T3485, +DNAP
 
-
** control 3: +template, +primer 4, +DNAP
 

Revision as of 13:42, 28 September 2012

PHIX174 Cell Free Expression Main project page
Previous entry      Next entry
Personal tools