User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/24: Difference between revisions
From OpenWetWare
Sean P Corum (talk | contribs) (Autocreate 2012/09/24 Entry for User:Sean_P_Corum/Notebook/PHIX174_Cell_Free) |
Sean P Corum (talk | contribs) m (→Entry title) |
||
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.=== | ||
* Gel electrophoresis results: | |||
** 1 μM primer mix = ~5 kb nicked DNA corresponding to ΦX174 genome | |||
** 10 μM primer mix = NO AMPLIFICATION\ | |||
* Ah ha! Since the 1 μM primer mix was diluted from the 10 μM primer mix, it is the case that overly concentrated primers inhibit PCR. | |||
* To optimize this: | |||
* 50 μL + 10% WP-PCR reaction: | |||
** 37 μL H<sub>2</sub>O | |||
** 5.5 μL 10X reaction buffer | |||
** 1.65 μL 3.2 nM ΦX174 template (~ 0.1 nM final) | |||
** 0.55 μL 25 mM dNTPs mix | |||
** 1.1 μL PfuUltra I DNA polymerase | |||
* Aliquot 5 × 9 μL: | |||
*# 1 μL water = 0 μM primer 4 | |||
*# 1 μL 12.5 μM primer mix = 1.25 μM primer 4 | |||
*# 1 μL 25 μM primer mix = 2.5 μM primer 4 | |||
*# 1 μL 50 μM primer mix = 5 μM primer 4 | |||
*# 1 μL 100 μM primer mix = 100 μM primer 4 | |||
* Cycling parameters: | |||
*# 95 °C 2 m | |||
*# 95 °C 30 s | |||
*# 58° C 30 s | |||
*# 72 °C 15 m | |||
*# Repeat 2-4 an additional 29 times for 30 total cycles | |||
*# 72 °C 30 m | |||
----------------- | |||
* 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 | |||
** control 4: +template, +primer 4 T3485, +DNAP | |||
** control 5: +template, -primers, +DNAP | |||
* Followed by: | |||
** (possible purification and then) DpnI digestion | |||
** PCR purification | |||
** Linking number adjustment by gyrase / topisomerase IV | |||
** PCR purification |
Revision as of 15:35, 24 September 2012
PHIX174 Cell Free Expression | <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> |
Hypothesis 2: Gene L is necessary for phage propagation.
|