User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/29: Difference between revisions
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< | * Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 and 1 μMM primer 4 (each) with variable elongation temperature, T<sub>e</sub>). | ||
*# 66 °C - 5.9 nM (2%) | |||
*# 68 °C - 10.4 nM (20%) | |||
*# 70 °C - 19.3 nM (15%) | |||
*# 72 °C - 49.4 nM (7%) | |||
*# 74 °C - 43.1 nM (6%) | |||
* Results ... | |||
* Next, testing variable annealing temperature: T<sup>e</sup> = 54, 56, 58, 60, 62 °C | |||
* 50 μL WP-PCR: | |||
** 31.2 μL H<sub>2</sub>O | |||
** 6.25 μL 2mM dNTPs (each) (0.25mM each final) | |||
** 5 μL 10X reaction buffer | |||
** 5 μL 10 μM primer 4 mix | |||
** 1.563 μL 3.2 nM ΦX174 template (0.1 nM final) | |||
** 1 μL PfuUltra II fusion HS DNA polymerase | |||
* Aliquot 5×10μL. | |||
* Cycling parameters: | |||
*# 95 °C 2 min | |||
*# 95 °C 20 s | |||
*# X °C 20 s, where X = 54, 56, 58, 60, 62 | |||
*# X °C 12 min | |||
*# Repeat 2-4 an additional 29 times = 30 cycles | |||
*# 12 °C hold | |||
--------- | |||
* Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 and 1 μMM primer 4 (each) with variable annealing temperature, T<sub>a</sub>). | |||
*# 54 °C - nM (%) | |||
*# 56 °C - nM (%) | |||
*# 58 °C - nM (%) | |||
*# 60 °C - nM (%) | |||
*# 62 °C - nM (%) | |||
* Results ... | |||
* Next, testing variable PCR cycling number N | |||
* 100 μL WP-PCR: | |||
** 62.4 μL H<sub>2</sub>O | |||
** 12.5 μL 2mM dNTPs (each) (0.25mM each final) | |||
** 10 μL 10X reaction buffer | |||
** 10 μL 10 μM primer 4 mix | |||
** 3.13 μL 3.2 nM ΦX174 template (0.1 nM final) | |||
** 2 μL PfuUltra II fusion HS DNA polymerase | |||
* Cycling parameters: | |||
*# 95 °C 2 min | |||
*# 95 °C 20 s | |||
*# X° C 20 s | |||
*# X °C 12 min | |||
*# 25 °C hold | |||
*# Repeat 2-5 an additional 44 times for N = 45 cycles, removing 10 μL samples for N = 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 | |||
*# 12 °C hold | |||
----------------- | |||
* Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 and 1 μM primer 4 (each) with variable PCR cycle number, N. | |||
*# 0 - nM (%) | |||
*# 5 - nM (%) | |||
*# 10 - nM (%) | |||
*# 15 - nM (%) | |||
*# 20 - nM (%) | |||
*# 25 - nM (%) | |||
*# 30 - nM (%) | |||
*# 35 - nM (%) | |||
*# 40 - nM (%) | |||
*# 45 - nM (%) | |||
* Results ... | |||
------------------- | |||
* Next, I will re-optimize primer concentration over the range 1mM/10^x, where x = 1, 2, 3, 4, 5, and ∞ (in reverse order, this is equivalent to 0, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM). 60 μL + 10% WP-PCR reaction: | |||
** 34.3 μL H<sub>2</sub>O | |||
** 6.6 μL 10X reaction buffer | |||
** 8.25 μL 2mM dNTPs (each) (0.25mM each final) | |||
** 2.063 μL 3.2 nM ΦX174 template (0.1 nM final) | |||
** 1.32 μL PfuUltra II fusion HS DNA polymerase | |||
* Aliquot 6×9 μL and add 10X primer range (0, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM). | |||
* Cycling parameters: | |||
*# 95 °C 2 min | |||
*# 95 °C 20 s | |||
*# 58 °C 20 s | |||
*# 72 °C ??? s | |||
*# Repeat 2-4 an additional 29 times = 30 cycles | |||
*# 72 °C 3 min | |||
*# 12 °C hold | |||
* Things that still need to be optimized: | |||
*# 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 | |||
* Here are the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable cycle number, N. | |||
*# 0 - nM ( %) | |||
*# 5 - nM ( %) | |||
*# 10 - nM ( %) | |||
*# 15 - nM ( %) | |||
*# 20 - nM ( %) | |||
*# 25 - nM ( %) | |||
*# 30 - nM ( %) | |||
*# 35 - nM ( %) | |||
*# 40 - nM ( %) | |||
* Optimizing N, the number of PCR cycles. 50 μL + 10% WP-PCR reaction: | |||
** 34.3 μL H<sub>2</sub>O | |||
** 6.88 μL 2mM dNTPs (each) (0.25mM each final) | |||
** 5.5 μL 10X reaction buffer | |||
** 5.5 μL ??? μM primer 4 mix | |||
** 1.719 μL 3.2 nM ΦX174 template (0.1 nM final) | |||
** 1.1 μL PfuUltra II fusion HS DNA polymerase | |||
* Aliquot 5×10μL and repeat 5 WP-PCR reactions for variable T<sub>a</sub>, where T<sub>a</sub> = 54, 56, 58, 60, and 62 °C. | |||
* Cycling parameters: | |||
*# 95 °C 2 min | |||
*# 95 °C 20 s | |||
*# T<sub>a</sub> °C 20 s | |||
*# 72 °C ??? s | |||
*# Repeat 2-4 an additional ??? times = ??? cycles | |||
*# 72 °C 3 min | |||
*# 12 °C hold | |||
* Next on the list: | |||
*# 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 17:15, 29 August 2012
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