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)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (RS300) ~10ng
Reaction 1(A&4) Reaction 2(3&2) Reaction 3(1&B) primer mix (1x) 2.5
Water 35

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 Amirnaassembly.jpg

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)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1" "2" "3") 1(parts 1&2); .5 (part 3)= 2.5 total
AB primer mix (1x) 2.5
Water 35

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

2 reactions (GFP, LTP)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1" "2" "3") 1(each)= 3 total
AB primer mix (1x) 2.5
Water 35

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)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
DNA (Parts "1" "2") 1(each)= 2 total
A2 primer mix (1x) 2.5
Water 35.5

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.