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

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

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.

Team Flavour

Miniprep of Miraculin and Brazzein Constructs

  • Protocol used was from the Qiagen Miniprep Kit
Nanodrop Specs
Name Concentration (ng/μL)
Miraculin 1 159.6
Miraculin 2 182.2
Brazzein 1 91.2
Brazzein 2 33.7

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

Digestion Reactions
pENTCUP2 NOSt NOSt+STOP B15
DNA 4 7 12 7
NEB Buffer 3 (10x) 2 2 2 2
diH2O 10 7 2 7
Xba1 1 1 1 1
Pst1 1 1 1 1
BSA (10x) 2 2 2 2
  • 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.

Digest Gel

Lane Contents
Lane 1 1KB Plus Ladder
Lane 2 pENTCUP2
Lane 3 NOSt
Lane 4 NOSt+STOP
Lane 5 B15 digested
Lane 6 B15 undigested


  • 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
Nanodrop Specs
Name Concentration (ng/μL)
pENTCUP2 16.5
NOSt 10.2
NOSt+STOP 0.4
B15 (backbone vector) 10.9
  • 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
pENTCUP2 NOSt NOSt+STOP Control
Insert 1 1 1 1
Vector 2 2 2 2
Buffer (10x) 5 5 5 5
Ligase 1 1 1 1
H2O 11 11 0 12

Team Fence

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.

1 2 3 4 5 6 7 8 9 10 11 12
Ladder Gal4 DBD 1 Gal4 DBD 2 Gal4 DBD 3 Barnase 1 Barnase 2 Barnase 3 Ladder


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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