IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/22

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Procedures
Today's objective is to take our PCR products from yesterday (LTP sense, Ger 3 sense, and Ger 3 antisense)and insert them into vector V0120 for E. coli. Our procedures were:
 * 1) Gel Extraction of LTP sense, Ger 3 sense, and Ger 3 antisense
 * 2) Purification of LTP sense, Ger 3 sense, and Ger 3 antisense
 * 3) Digest of LTP sense, Ger 3 sense, and Ger 3 antisense with Xba and Pst restriction enzymes
 * 4) Purification of digested allergens LTP sense, Ger 3 sense, and Ger 3 antisense
 * 5) Digest of V0120 with Xba and Pst restriction enzymes
 * 6) Gel Electrophoresis of the digested vector
 * 7) Gel Extraction and Purification of Vector
 * 8) Ligation of V0120 and inserts for each LTP sense, Ger 3 sense, and Ger 3 antisense

Results
'''1. Gel Extraction of LTP sense, Ger 3 sense, and Ger 3 antisense 2. Purification of LTP sense, Ger 3 sense, and Ger 3 antisense'''

Obtained 2.6, 7.8 and 7.9 ng/μL of LTP sense, Ger 3 sense, and Ger 3 antisense, respectively, after ourgGel extraction and purification. These are relatively small concentrations. These tubes of DNA will be digested and then purified again.

3. Digest of LTP sense, Ger 3 sense, and Ger 3 antisense with Xba and Pst restriction enzyme

4. Purification of digested allergens LTP sense, Ger 3 sense, and Ger 3 antisense

Obtained 1.5, 4.3 and 0.5 ng/μL of digested LTP sense, Ger 3 sense, and Ger 3 antisense, respectively. The drop in concentration is attributed to loss of DNA through the purification process.

5. Digest of V0120 with Xba and Pst restriction enzymes 6. Gel Electrophoresis of the digested vector

The properly digested vector is 3000bps long, and rests between two bands on the gel -- the uncut or one cut vector and the removed inserts.

7. Gel Extraction and Purification of Vector Nanodrop of digested vector yielded 25.2 ng/μL.

8. Ligation of V0120 and inserts for each LTP sense, Ger 3 sense, and Ger 3 antisense

Ligated vectors were put on ice and set aside for tomorrow, where they will be transformed into competent turbo E. coli, which will be grown in plates of agar. Colonies that grow should contain closed vector plasmid, which contains ampicillin resistance and inserts.

GAL4DBD Miniprep



 * pipetted 4 ml from each overnight cell culture from colonies 1-5 into a 15 ml conical centrifuged at 4400 rpm for 6 minute
 * remaining overnight cell cultures were placed in fridge
 * decanted LB+amp, resuspended cells in 250 μL P1 buffer
 * contents transferred to eppendorfs
 * 250 μL of P2 buffer was added per tube, the tubes were inverted 4-6 times
 * 350 μL N3 buffer added to each, tubes inverted 4-6 times
 * centrifuged for 10 min at 13,000 rpm, supernatant collected ad transferred to QIAprep spin columns
 * centrifuged for 30-60 seconds, flow through discarded
 * 0.5 ml buffer PB added and centrifuged for 30-60 seconds, flow through discarded, spun columns for an additional minute
 * QIAprep columns put into new eppendorfs
 * 50 μL buffer EB added to columns, let stand for 1 minute
 * centrifuged for 1 minute at 13,000 rpm
 * tubes labeled "GAL4" plus the specific colony number

PCR
PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)

1=95°C for 10:00

2=95°C for 00:15

3=50°C for 00:30

4=72°C for 01:30

5=steps 2-4 X 29

6=72°C for 10:00

7=4°C for ∞



PCR for E10 LacI
 * above PCR was repeated without O2 so as to finalize our E10 biobrick for use
 * PCR was performed using the standard proportions of reagents
 * The above settings on the PCR machine were used

To Make Agarose Gel
 * 150 mL TAE buffer combined with 1.5 g agarose in beaker
 * solution microwaved until agarose disolved
 * 7.5 μL Ethidium Bromide added after beaker was no longer steaming
 * solution poured into clean gel mold and gel was left to solidify
 * E10 PCR product from the morning was consolidated into one tube,
 * final volume =90μL PCR product + 18μL 5X loading dye =110μL

DNA was extracted from gel by slicing out the bands and DNA was purified using the QIAquick Gel Extraction Kit, following protocol specific to using a centrifuge

see QIAquick spin handbook

Innoculations
Colonies from the following cultures were innoculated and set to shake at 37°C overnight:
 * PMT413
 * PMT316
 * firefly luciferase
 * GFP (mut)
 * cre recombinase
 * lox66
 * lox71


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