User:Lu Wang/Notebook/Team Allergy/2010/06/28

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=Procedures=

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.

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

=Results=

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.


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