User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/24

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m (Hypothesis 2: Gene L is necessary for phage propagation.)
m (Hypothesis 2: Gene L is necessary for phage propagation.)
Line 13: Line 13:
* 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.
* 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:
* To optimize this:
-
* 50 μL + 10% WP-PCR reaction:
+
* 0.45 mL 90% ΦX174 WP-PCR Mix (reusable)
-
** 37 μL H<sub>2</sub>O
+
** 370 μL H<sub>2</sub>O
-
** 5.5 μL 10X reaction buffer
+
** 50 μL 10X reaction buffer
-
** 1.65 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
+
** 15 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
-
** 0.55 μL 25 mM dNTPs mix
+
** 5 μL 25 mM dNTPs mix
-
** 1.1 μL PfuUltra I DNA polymerase
+
** 10 μL PfuUltra I DNA polymerase
-
* Aliquot 5 × 9 μL:
+
* Aliquot 5 × 4.5  μL:
-
*# 1 μL water = 0 μM primer 4
+
*# 0.5 μL water = 0 μM primer 4
-
*# 1 μL 12.5 μM primer mix = 1.25 μM primer 4
+
*# 0.5 μL 12.5 μM primer mix = 1.25 μM primer 4
-
*# 1 μL 25 μM primer mix = 2.5 μM primer 4
+
*# 0.5 μL 25 μM primer mix = 2.5 μM primer 4
-
*# 1 μL 50 μM primer mix = 5 μM primer 4
+
*# 0.5 μL 50 μM primer mix = 5 μM primer 4
-
*# 1 μL 100 μM primer mix = 100 μM primer 4
+
*# 0.5 μL 100 μM primer mix = 10 μM primer 4
* Cycling parameters:
* Cycling parameters:

Revision as of 19:08, 24 September 2012

PHIX174 Cell Free Expression Main project page
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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:
  • 0.45 mL 90% ΦX174 WP-PCR Mix (reusable)
    • 370 μL H2O
    • 50 μL 10X reaction buffer
    • 15 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
    • 5 μL 25 mM dNTPs mix
    • 10 μL PfuUltra I DNA polymerase
  • Aliquot 5 × 4.5 μL:
    1. 0.5 μL water = 0 μM primer 4
    2. 0.5 μL 12.5 μM primer mix = 1.25 μM primer 4
    3. 0.5 μL 25 μM primer mix = 2.5 μM primer 4
    4. 0.5 μL 50 μM primer mix = 5 μM primer 4
    5. 0.5 μL 100 μM primer mix = 10 μM primer 4
  • Cycling parameters:
    1. 95 °C 2 m
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 72 °C 15 m
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 72 °C 30 m

  • Next on the list:
    1. DpnI digestion (w/ and w/o PCR purification)
    2. 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

Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • I minprepped pBEST-Pr-MGapt-UTR1-deGFP-T500 from last weeks frozen tubes of midi-culture using the Sigma kit.
  • I reduced volume from 1.2 mL to ~100 μL by spinvac, and then I purified the DNA by PCR Purification Kit.
  • I verified the plasmid DNA by DD with SphI and NcoI, which was expected to yield 3102 bp and 139 bp dsDNAs.
  • Gel electrophoresis showed a band at ~3000 bp, though the 139 bp band was not visible on this gel (neither was the 100 bp ladder). Uncut plasmid DNA traveled farther, and there were two higher order plasmid states, which disappeared in the cut DNA, as expected.
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