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

Preparation of Reaction Solutions

1. gBlocks

   2\(\mu\)l of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20\(\mu\)l to make 1ng/\(\mu\)l^-1

2. Primers

   2\(\mu\)l of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20\(\mu\)l to make 10\(\mu\)M reaction solutions.(The 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.

25\(\mu\)l reactions were run, with the volume breakdown by component being:

Component	Volume/\(\mu\)l	Final conc/nM
Q5 HF Master Mix	12.5	-
10\(\mu\)M Forward Primer	1.25	500
10\(\mu\)M Reverse Primer	1.25	500
1ng/\(\mu\)l-1	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 here.

** 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 DNA Melting	98	30
25-35

* 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.

Gel Electrophoresis of PCR-Amplified gBlocks

General guidelines for agarose preparation

Fragment size	Agarose gel w/v %	Mass of agarose in 200ml 0.5x TBE / g
>3kb	0.8	1.6
<1kb	2	4
In between	1	2

Agarose Preparation Protocol

1. Heat 2g agarose in 200ml 0.5x TBE for 2 minutes under full power in the microwave (use a 500mL Duran bottle, and place a weighing boat underneath it to prevent the causing of a mess in the event the mixture boils over; DO NOT fully tighten the Duran cap).
2. Check if the agarose has been fully dissolved. Heat it further if gel strands are visible.
3. Leave the agarose solution to cool at 50℃ for 20 minutes.
4. Pour agarose onto gel plate in a setting tray with appropriately-sized combs already fixed onto it, and leave for 20 minutes to let it set.
5. When the agarose has set, remove the combs and transfer the gel plate from the setting tray to the electrophoresis chamber.
6. Flood the gel plate with 0.5x TBE buffer up until right above the top of the wells.
7. The gel should be positioned such that the positive (red) electrode is on the far side of the gel from the wells, as the negatively-charged DNA will migrate towards the positive electrode.

DNA preparation:

The contents of the PCR tubes need to be stained with a loading dye to help visualize its migration.

To each 25\(\mu\)l of content in each PCR tube, 10\(\mu\)l of blue loading dye was added.