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
- PCR reactions of allergen panel with primers (A,IV), (III,II), and (I,B)
- Gel Electrophoresis to find parts
- Gel Purification for Parts
Ligating Parts Together
- Three in one sewing PCR
- 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.
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'
|