IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/28

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Team Allergy
Today, our objective is to finish the construct for amiRNA so that it will be ready for insertion into V0120. We will run a series of PCR reactions to replace the existing interference sequence with our own sequence using a series of primers. Then, we will sew the small pieces of DNA together in another PCR reaction.

Procedures
Creating Parts of amiRNA
 * 1) PCR reactions of allergen panel with primers (A,IV), (III,II), and (I,B)
 * 2) Gel Electrophoresis to find parts
 * 3) Gel Purification for Parts

Ligating Parts Together
 * 1) Three in one sewing PCR
 * 2) Two in one sewing PCR, followed by another two in one PCR

Creation of Parts 1,2,3 (Primers A&4; 3&2; 1&B)
Annealing Temp: (LTP & Bet)60C (GFP)69C Extension Temp: 15 sec

Total of nine reactions: three pieces for each GFP, Betv1, and LTP.

9 (3*3) reactions (GFP; Bet; LTP)

Nanodrop

Concentrations:

LTP 1: 5.4 ng/uL 2: 5.5 ng/uL  3: 1.2 ng/uL GFP 1: 17 ng/uL 2: 6.6 ng/uL  3: 15.1 ng/uL Bet 1:7.4 ng/uL 2: 29.6 ng/uL 3: 34.3 ng/uL

Gel Electrophoresis

PCRs A& B worked: 1 (ladder); 2(LTP 1+2); 3 (Bet 1+2); 4(GFP 1+2); 5(Ladder); 6(LTP 1,2,3); 7(Be 1,2,3); 8(GFP 1,2,3); 9(LTP 1,2,3(using .5ul)); 10(GFP 1,2,3 (using .5 uL))

Lanes 2-4 should be ~a little less than 450 bp and 6-10 should be ~450bp

Ligation of Parts
A: Mix of Parts 1,2,3 w/ primers A&B (Three in one PCR, or simultaneous PCR)

Annealing Temp: 60C Extension Time: 15 sec

3 reactions (GFP; Bet; LTP)

Concentrations: GFP 47.3 ng/uL; LTP 79.7 ng/uL; Bet 80.4 ng/uL

2 reactions (GFP, LTP)

Concentrations: GFP 51.5 ng/uL; LTP 67.3 ng/uL

B: Step 1: Mix of Parts 1&2 w/ Primers A&2,

Step one of two piece PCR

3 reactions (GFP; Bet; LTP)

Concentrations: GFP 45.9 ng/uL; LTP 17.8 ng/uL; Bet 24.2 ng/uL

Bands actually not what we are looking for (should be a little larger)

Step Two of Two Piece PCR

Sewing together parts from step one with part number three of each allergen with primers A and B.

Result was successful -- had bands that were ~500bps in length.

Miniprep of Miraculin and Brazzein Constructs

 * Protocol used was from the Qiagen Miniprep Kit

DTL (Digestion, Ligation, Transformation) of The Big Three (pENTCUP2, NOSt, NOSt + STOP)

 * Digestions were left for 1:30 at 37°C
 * 2.2 μL of DNA Loading Buffer were added to each reaction and loaded onto a 1% Agarose gel (TAE buffer). Gel was ran at 125 V for 30 min.




 * The undigested B15 in lane 6 appears as two bands on account of supercoiling of the B15 plasmid.
 * Bands in lanes 2 - 5 were cut out and purified using a Qiagen Gel Purification kit
 * The gel purification was done with 300 μL Buffer PB and 400 μL Buffer QG


 * Note the very low concentration of NOSt+STOP. It is uncertain as to what caused such a concentration, but transformation proceeded anyways with max volume
 * The ligation reactions were preformed at at 2:1 (insert to backbone) Molar Ratio
 * The sole exception being the NOSt+STOP ligation, in which the lack of product forced us to use 1.8 ng DNA


 * Ligation reactions were preformed as per the Silver Lab ligation protocol (with the differences in concentration and ratio made as per the table above).
 * Transformations to E. Coli were preformed as per the Silver lab transformation protocol with ampicillin plates.
 * Plates were left overnight.

B21 Innoculation
Inoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone.

PCR of Gal4DBD and Barnase, attempt 2
Ran 3 tubes of each for a total of 6. 7x Mastermix:
 * 14μL DNTP
 * 3.5μL Polymerase
 * 28μL 5x Buffer
 * 73.5μL DH2O

Each tube contained:
 * 1μL Fwd primer
 * 1μL Rev Primer
 * 1μL Minipreped sample
 * 17μL Mastermix

Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program

Gel of Gal4 DBD and Barnase from second PCR attempt
Ran 1.2% E-gel of the 6 PCR product tubes.



QIAQuick Purification of PCR Product
Because colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.
 * as there was about 25μL of PCR product in the two together, 125μL of PB buffer was added (5x the concentration of the PCR product)
 * pipetted the mix into a QIAquick spin column, spun for 30 seconds at 13,000 rpm
 * discarded flow-through
 * added .75mL PE buffer to the column, spun for 30 seconds
 * discarded flow-through, and spun again for 1 minute
 * moved the column to a fresh eppendorf tube
 * added 50 μL EB buffer to the column, spun for 1 minute
 * labeled the eppendorf tube 'Barstar PCR purified'


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