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 200\(\mu\)g 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

25µl reactions were run according to the PCR protocol here

* 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 PCR program was run.

* 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

An agarose gel was prepared according to the agarose prepartion protocol.

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 from 22/06)

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.

The gel was then stained and the bands were visualized; protocol

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

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 Quantification using NanoDrop

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:

Sample	Concentration/ng\(\mu\)l
C	11.3
D	3.5
E	0.8
H	0.9
J	1.6
L	7.7
N	3.6
pSB-1C3	1.6

Ligation of gBlock Sequences with pSB-1C3 Backbone

Ligation was completed; protocol

Preparation of Competent E. coli Cells

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℃.

Re-PCR of DNA Sequences

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.