User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/26: Difference between revisions
From OpenWetWare
Sean P Corum (talk | contribs) m (→Entry title) |
Sean P Corum (talk | contribs) |
||
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 showed the following over the rest of the primer 4 mix range: | |||
** Nothing the the column of -primer control. | |||
** A strong band at > 5kb at 0.5 μM primer (band 1), growing weaker as primer concentration increased, and disappearing for the 4 and 8 μM primer columns. Band 1 = amplified, nicked ΦX174 genome. | |||
** A weak band at < 100 bp (band 2) at 0.5 μM primer (band 2), growing stronger as primer concentration increased. Band 2 = ssDNA primers. | |||
** A weak band at < 100 bp (band 3) > band 2, not apparent until 1 μM (nothing at 0.5 μM) and growing stronger with increasing primer concentration. Band 3: hybridized ssDNA primers. | |||
* Therefore, as I anticipated, too much primers inhibited PCR due to primer hybridization of complementary primers used for WP-PCR. | |||
* 0.5 μM should be used for WP-PCR amplification of ΦX174 genome. I finalized the WP-PCR protocol. Other long WP-PCRs should use between 0.1 and 1 μM primer, with the optimal primer concentration being found for each new primer/template combination. | |||
* Final WP-PCR protocol: | |||
** | |||
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2) | |||
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2) | |||
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2) | |||
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2) | |||
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2) | |||
** 0.5 μM: stong ~≥ 5 kb (amplified ΦX174 genome), weak ~>100 bp (band 2) | |||
* Given these results, here's what I think is happening. At too high primer concentration, the ssDNA primers, being complementary strands of WP-PCR, do not unbind, inhibiting PCR. | |||
* I will do one more test over a primer 4 mix of 0, 0.5, 1, 2, 4, and 8 μM. | |||
* Aliquot 6 × 4.5 μL ΦX174 WP-PCR mix (1X reaction buffer, .25 mM dNTPs mix, PfuUltra I DNAP, ~0.1 nM ΦX174 template) | |||
*# 0.5 μL 5 water | |||
*# 0.5 μL 5 μM primer 4 mix | |||
*# 0.5 μL 10 μM primer 4 mix | |||
*# 0.5 μL 20 μM primer 4 mix | |||
*# 0.5 μL 40 μM primer 4 mix | |||
*# 0.5 μL 80 μM primer 4 mix | |||
* WP-PCR 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 | |||
===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.=== | ===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.=== | ||
Revision as of 13:41, 27 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.
Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.
|