Team:Oxford/Notebook/Week1

Team Oxford


Week 1




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


Setting up Agarose Gel for Electrophoresis for 22/06 PCR Products


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 4

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 25µl of content in each PCR tube, 10µl of blue loading dye was added.
23/06/2015 George, Silas,
Mabel, June

Gel Electrophoresis of PCR-Amplified gBlocks (continued from 22/06)


For reference - gBlock sizes:

Label Construct Size / bp
A LasR Holin 1763
B LasR sfGFP 1829
C Lsr sfGFP 910
D Lsr Holin 862
E DNase DsbA 646
F DspB YebF 1525
G DspB 1174
H MccS 448
I DspB Fla 1336
J DspB DsbA 1228
K Art-175 DsbA 1012
L Art-175 YebF 1309
M Art-E 644
N Art-175 Fla 1120

Lane 1: 10µl of 2-Log Ladder (https://www.neb.com/products/n0550-quick-load-purple-2-log-dna-ladder-0-1-10-0-kb).
Lanes 2-15: 20µl of DNA + loading dye mixture prepared in 22/06.

A potential difference of 120V was applied across the electrodes (the higher the voltage, the faster the gel will run but the poor the separation will be; DNA separation is typically done in the 120-140V range) for 80 minutes. As long as DNAs have passed ⅔ of the column or purple dye have passed purple area of the gel, the gel is ready to get into the EtBr.

Stain gel using ethidium bromide according to the following protocol:

    1. Pick up the gel keeping it flat
    2. Slide carefully into a vat of ethidium bromide
    3. Set the vat to gentle shaking for 30 minutes
    4. Pick up the gel using a spatula and rinse off the toxic ethidium bromide
    5. Visualise using transilluminator

Ethidium bromide stains DNA such that it fluoresces with an orange colour under UV light.


Visualizing Separated DNA in Agarose - UV Transilluminator

    1. Place the gel on the transilluminator stage and adjust stage height appropriately.
    2. Set the transilluminator using the GeneSnap program such that the light emitted is UV (instead of white light) and the software filter is configured to pick up EtBr fluorescence.
    3. Adjust the contrast such that the bands can be clearly seen.
    4. Adjust the focus using the focusing rings to sharpen the image.
    5. Save the image in the naming format “dd_mm_yy” to Disk C: → Lab users → iGEM in .sdg file format, and additionally export it as a .tif file.


Results: