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

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

Polymerase Chain Reaction Set-Up

Mabel pipetting

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

Leon pouring the gel

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.

Gel Electrophoresis of PCR-Amplified gBlocks Continued

gBlock sizes for reference:

Lane 1: 10\(\mu\)l of 2-Log Ladder

Lanes 2-15: 20\(\mu\)l of DNA and loading dye mixture prepared on 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 DNA has 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:

PCR Results

The 7 bands corresponding to expected DNA sizes were excised using a razor blade for cleaning and extraction. The other sequences, along with J which only showed a very weak band, will be re-PCRed under modified conditions at a later date.

The excised gel chunks were transferred to centrifugation tubes.

Extraction of DNA(PCR product) from Agarose Gel

The extraction was performed using the E.Z.N.A. Gel Extraction Kit made by Omega Biotek, according to the Spin Protocol.

The excised agarose chunks needed to be dissolved in a minimum of 1mL of XP2 Binding Buffer per gram of gel. The heaviest chunk of gel weighed 0.16g and as such 160µl of buffer was added to each tube.

* As the tubes were spun with their lids open, they were placed such that lids pointed away from the direction of spinning.

Restriction Digest of Extracted PCR product

Restriction digest was performed using EcoRI-HF (5’) and SpeI (3’) restriction enzymes.

NEB’s Double Digest Finderwas invoked and it was determined that CutSmart Buffer would be used for the digest.

The buffer was completely defrosted and shaken before use.

There is a recommended digestion protocol on NEBCloner. 50µL reaction mixtures were set up with component volumes as recommended, except for the DNA where all 30µL of the extraction mixture (in elution buffer) were used in the digest. There would be up to 50ng of DNA in each tube of extraction mixture (from the gel bands).

Incubation at 37℃ was also done for 2 hours (ThermoMixer program 3) instead of the recommended 5-15 minutes, with shaking at 300rpm.

Restriction Enzyme Digest of the iGEM HQ linearised pSB-1c3

125ng of pSB-1C3 was dissolved in 5mL Milli-Q water

The digest was performed using the modified NEBCloner protocol:

* We did not add phosphatase because it is assumed that with different sticky ends the vector cannot religate

DNA ‘Cleanup’ using EZNA enzymatic reaction kit

Protocol can be found at the end of EZNA gel extraction kit

Upon completion of restriction digest incubation, the gBlocks and the plasmid backbones were purified again using the E.Z.N.A. Gel Extraction Kit. PCR products and plasmid backbones need to be purified in order to remove remaining impurities including agarose gel and restriction enzymes. At the elution step, the gBlock inserts were eluted using 30µL elution buffer whereas the plasmid backbone was eluted using 50µL of it.

DNA ‘Cleanup’ using EZNA enzymatic reaction kit

1\(\mu\)L water and tissue were first used to clean the stage. A blank reading was made using 1\(\mu\)L of elution buffer, and 1\(\mu\)L of each sample was measured for concentration:

Ligation of gBlock Sequences with pSB-1C3 Backbone

T4 DNA Ligase was defrosted before use, kept on a freezing block when on the bench, and was the last to be added to the reaction mixture.

The 49\(\mu\)L of plasmids were split into seven 7\(\mu\)L portions (~ 125ng / 7 = 18ng), matching up with ~ 50ng of each gBlock to give a desirable vector : insert ratio of almost 1 : 3.

The component volumes are:

The reaction mixture was incubated at 16℃ overnight.

A sample of E. coli DH5ɑ stored at -80℃ was defrosted and inoculated in 5mL of LB in a 125mL conical flask (volume of LB 10% of flask volume so as to achieve sufficient aeration). The culture was left to grow overnight at 37℃.

Two sets of sequences A,B,F,G, I, J, K, and M were PCRed using the same protocol as 22/06, with one set using a 55℃ annealing temperature and another using 50℃ annealing temperature.