Difference between revisions of "Team:Oxford/Notebook/Week1"

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       <p class="text">* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s online tool at http://tmcalculator.neb.com/#!/.<br>
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       <p class="text">* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s <a href="http://tmcalculator.neb.com/#!/">online tool</a>.<br>
  
 
** Add components in order of decreasing volume for maximum ease-of-pipetting.<br>
 
** Add components in order of decreasing volume for maximum ease-of-pipetting.<br>

Revision as of 13:14, 2 July 2015

Team Oxford




Date Researcher(s) Content
22/06/2015

Whole Team
(Introductory Session)

Preparation of Stock Solutions


1. gBlocks

The gBlocks ordered from IDT arrived in the form of vials of 200ng solid DNA powder.

(refer to BioBricks page for information on DNA sequences)

The gBlocks were made into 10ng/µl stock solutions in Milli-Q water for storage:

mass / ng conc / ngµl-1 final volume / µl
200 10 20
2. Primers

The forward and reverse primers ordered from IDT came in 32.4nmol and 34.3nmol of solid respectively.

(Sequences: Forward - CTTTTTTGCCGGACTGC; Reverse - ATGATTTCTGGAATTCGC)

The primers were made into 100µM stock solutions in Milli-Q water for storage:

amt / 10-9 mol conc / 10-6 M final volume / 10-6 L
32.4 100 324
34.3 100 343


Preparation of Reaction Solutions


1. gBlocks

2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 1ng/µl-1 reaction solutions.


2. Primers

2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 10µM reaction solutions. (These solutions are labelled as “Prefix primer” and “suffix primer” in eppendorf tubes in the fridge)


Polymerase Chain Reaction Set-up


The protocol for running a PCR using NEB’s Q5 High-Fidelity 2X Master Mix can be found here.
25µl reactions were run, with the component breakdown by volume being:


Component Volume/µl Final concentration/nM
NEB Q5 HF Master Mix 12.5 -
10µM Forward Primer 1.25 500
10µM Reverse Primer 1.25 500
1ng/µl-1 gBlock 1.0 -
Milli-Q Water 9.0 -

* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s online tool.
** Add components in order of decreasing volume for maximum ease-of-pipetting.
*** When reaction mixture is being made up, all components as well as the mixture itself are to be kept on ice, as the Master Mix is a high-fidelity polymerase that will recognize the primers as being incorrectly base-paired and be able to hydrolyse the primers if kept at room temperature.
**** Use Q5 enzyme in the cold room to avoid defrosting and freezing the original stock of Q5 enzyme. This could decrease the activity of Q5 enzyme. Bring ice bucket to the cold room to bring Q5 into the bench.
***** Make sure that the primer and small amounts of DNA and primer doesn’t stick onto the side of the tube or the tip.


The reaction mixture tubes were positioned in an Eppendorf Mastercycler nexus X2 and the following PCR program was run:


Cycle(s) Step Temp / ℃ Time / s
1 Initial template DNA melting 98 30
30 DNA Meting 98 10
Primer Annealing 55 20
Sequence Extension 72 60
1 Final Extension 72 120
1 Hold 4-10 -

* DNA denaturation can be performed at 98℃ because of the high thermal stability of the Q5 polymerase
** A PCR takes 20-30 seconds to extend a sequence by 1kb, and since our longest sequence is ~2kb the extension time was determined to be 60s per cycle