User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/21: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 17: Line 17:
** 100 μM AS primer - 60 μM (18%); this needs to be repeated
** 100 μM AS primer - 60 μM (18%); this needs to be repeated


* The last three data points satisfies the linear equation product = 4.99x+67.5 with R<sup>2</sup> = 0.9997. The fact that this is growing linear indicates that the WP-PCR is outside of exponential PCR. I hypothesize that the elongation time is too short. So that is I will optimize this next, over elongation times of 0.5, 1, 2, 5, and 10 min, with primer concentration fixed at 1 μM.
* The last three data points satisfies the linear equation product = 4.99x+67.5 with R<sup>2</sup> = 0.9997. The fact that this is growing linear indicates that the primers are the limiting component in WP-PCR (as primers typically are in WP-PCR). Next, I will optimize elongation time.
 
* 50 μL WP-PCR reaction.
* 50 μL WP-PCR reaction.
** 39.6 μL H<sub>2</sub>O
** 39.6 μL H<sub>2</sub>O
Line 35: Line 34:
*# 72 °C 3 min
*# 72 °C 3 min
*# 12 °C hold
*# 12 °C hold
* Things that still need to be optimized:
*# After primer concentrations have been corrected, vary primer concentration by 0, 1E2, 1E3, 1E4, 1E5, 1E6 nM = 1 mM
*# T<sub>a</sub> = 54, 56, 58, 60, 62 °C
*N cycles = 0, 10, 20, 25, 30, 35, 40, 50
* After that, tasks include characterizing effects of:
*# parallel PFU ligation
*# 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 14:48, 21 August 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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Hypothesis 2: Gene L is necessary for phage propagation.

  • Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable 36-mer primer concentration. ΦX174 1 nM template only control gave ~0 nM w/ inconsistent readings (not really a problem).
    1. 0 μM - 0.36 nM inconsistent
    2. 1 μM - 0.31 nM inconsistent
    3. 2 μM - 78 nM
    4. 5 μM - 92 nM
    5. 10 μM - 117 nM
    • 100 μM S primer - 39 μM (0.8%)
    • 100 μM AS primer - 60 μM (18%); this needs to be repeated
  • The last three data points satisfies the linear equation product = 4.99x+67.5 with R2 = 0.9997. The fact that this is growing linear indicates that the primers are the limiting component in WP-PCR (as primers typically are in WP-PCR). Next, I will optimize elongation time.
  • 50 μL WP-PCR reaction.
    • 39.6 μL H2O
    • 5.5 μL 10X reaction buffer
    • 6.88 μL 2mM dNTPs (each) (0.25mM each final)
    • 5.5 μL primer 4 mix (each)
    • 1.562 μL 3.2 nM ΦX174 template (0.1nM final)
    • 1 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 5×10 μL.
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. 72 °C X min (0.5, 1, 2, 5, 10)
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 72 °C 3 min
    7. 12 °C hold
  • Things that still need to be optimized:
    1. After primer concentrations have been corrected, vary primer concentration by 0, 1E2, 1E3, 1E4, 1E5, 1E6 nM = 1 mM
    2. Ta = 54, 56, 58, 60, 62 °C
  • N cycles = 0, 10, 20, 25, 30, 35, 40, 50
  • After that, tasks include characterizing effects of:
    1. parallel PFU ligation
    2. DpnI digestion (w/ and w/o PCR purification)
    3. 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, Ta, 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
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Sean_P_Corum/Notebook/PHIX174_Cell_Free/2012/08/21&oldid=619272"