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Revision as of 13:56, 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> -