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

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                                     The reaction mixture tubes were positioned in an Eppendorf Mastercycler nexus X2 and the following PCR program was run:
 
                                     The reaction mixture tubes were positioned in an Eppendorf Mastercycler nexus X2 and the following PCR program was run:
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                                             <th>Cycle(s)</th>
 
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                                             <td>25-35</td>
 
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                                 <h4>Gel Electrophoresis of PCR-Amplified gBlocks</h4>
 
                                 <h4>Gel Electrophoresis of PCR-Amplified gBlocks</h4>
 
                                 <p>General guidelines for agarose preparation
 
                                 <p>General guidelines for agarose preparation
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                                             <th>Fragment size</th>
 
                                             <th>Fragment size</th>

Revision as of 14:28, 13 August 2015

Notebook

Week 1

Week 1 - Day 1

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/ng\(\mu\)l-1 final volume\(\mu\)l
    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/10-9mol conc/10-6M final volume/10-6L
    32.4 100 324
    34.3 100 343

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.