Team Flavor
Miniprep of STOP + V0120 part
Nanodrop O/D's:
STOP V0120 1-1: 134.4 ng/μL
STOP V0120 1-2: 98.7 ng/μL
STOP V0120 2-1: 55.2 ng/μL
STOP V0120 2-2: 121.8 ng/μL
DTL of StrepII+Mira/Brazz with STOP codon
- To insert a STOP codon to the end of our Mira/Brazz StrepII constructs, we used the STOP+V0120 BioBrick as our vector (EcoR1/Xba1 digest) and our Miraculin/Brazzein construct as our insert (EcoR1/Spe1 digest)
- Construct DNA used was from Miniprep 1 (Box 4)
- ~1μg of DNA was digested in each reaction
Digestion Reactions
|
Mira Strep N |
Mira Strep C |
Brazz Strep N |
Brazz Strep C |
STOP+V0120 BioBrick
|
diH2O
|
15 |
14.5 |
13.5 |
11.5 |
6
|
Green FD buffer (10x)
|
2 |
2 |
2 |
2 |
2
|
DNA
|
1 |
1.5 |
2.5 |
4.5 |
10
|
EcoR1
|
1 |
1 |
1 |
1 |
1
|
Xba1
|
- |
- |
- |
- |
1
|
Spe1
|
1 |
1 |
1 |
1 |
-
|
Digestion of Mira/Brazz+Strep and STOP codon BioBrick
- Circled bands were cut and gel purified
Miraculin + StrepII = 730 bp
Brazzein + StrepII = 230 bp
STOP + V0120 = 3200 bp
- 1kb Plus Ladder
- Miraculin C Strep E/S
- Miraculin N Strep E/S
- Brazzein C Strep E/S
- Brazzein N Strep E/S
- STOP + V0120 E/X
- 1kb Plus Ladder
Gel Purification Specs
Mira N StrepII E/S: 3.0 ng/μL
Mira C StrepII E/S: 2.8 ng/μL
Brazz N StrepII E/S: 3.4 ng/μL
Brazz C StrepII E/S: 1.3 ng/μL
STOP+V0120 BB E/X: 7.1 ng/μL
Ligation of Mira/Brazz+Strep and STOP codon BioBrick
Ligation Reactions
|
Mira StrepII N + STOP w/ v0120 BB |
Mira StrepII C + STOP w/ v0120 BB |
Brazz StrepII N + STOP w/ v0120 BB |
Brazz StrepII C + STOP w/ v0120 BB |
Control STOP w/ v0120 BB
|
diH2O
|
4.5 |
4.5 |
9 |
5 |
12
|
T4 DNA ligase buffer (10x)
|
2 |
3 |
2 |
2 |
2
|
DNA Insert
|
7.5 |
7.5 |
2 |
6 |
0
|
DNA Backbone
|
5 |
5 |
6 |
6 |
5
|
T4 DNA ligase
|
1 |
1 |
1 |
1 |
1
|
Transformation
- Transformed 5 μL ligation mix with 15 μL TURBO e. coli cells
- Plated on LB + Amp plates and left in 37°C incubator overnight
Team Fence
PCR of the arabidopsis promoters with the newly extracted DNA and the older DNA.
FAIL
Arp2 promoter PCR failed.
Exp2 promoter PCR failed.
- resuspend DNA in approximately 200μL molecular grade pure water
- add 1 volume phenol chloroform
- when pipetting the phenol chloroform be sure to pipette from closer to the middle level of the liquid as the top two centimeters do not contain what we want
- centrifuge at top speed for 5 minutes
- collect the aqueous top layer and transfer to a clean microcentrifuge tube
- the remaining liquid is toxic and must be carefully disposed of in the "toxic waste" bottle in the fume hood
- add 1 volume phenol chloroform
- centrifuge at top speed for 5 minutes
- collect the aqueous top layer and put into a fresh tube
- add three volumes 100% ethanol and 1/10 volume sodium acetate
- allow solution to incubate on ice for 15 minutes
- centrifuge at top speed for 10 minutes
- pipette off the aqueous solution leaving a pellet of DNA in the bottom of the tube
- wash in 500μL 70% ethanol
- mix tube by inverting
- centrifuge again for 10 minutes at top speed
- air dry pellet by leaving tube open on bench (not on ice) til dry
- after pellet has dried resuspend DNA in desired volume of H2O or buffer EB
- presence of DNA can be confirmed by running a small sample on a gel
THE END
NLS Ligations
Ligation of NLS.Serine
- 2.5μL Backbone
- 1.5μL of 1/100 dilution (1.5ng NLS.Serine)
- 6μL H2O
- 10μL 2x quick ligase buffer
- 1μL quick ligase
B21 control
- 2.5μL B21 Backbone
- 7.5μL DH2O
- 10μL 2x Quick Ligase Buffer
- 1μL Quick Ligase
Transformation
- 1μL NLS lig into TOP10 cells
- Chilled on ice for 30 mins
- Heat shocked at 42°C for 30 seconds
- Put on ice for 2 mins
- Added 170μL SOC medium
- Set to shake in incubator for 30 mins
- Streaked on LB+amp plates, incubated overnight
Yeast backbone gel purified nanodrops
Sample
|
Concentraion in ng/μL
|
260/280
|
31 (1)
|
8.5 |
3.00
|
31(2)
|
10.2 |
2.07
|
32(3)
|
11.1 |
2.31
|
52(4)
|
28.5 |
1.83
|
Team Allergy
- Genomic DNA extraction
- PCR for allergen panel from genomic DNA
- Grew up cultures of transformations from yesterday (LTPS+intron; GerS+intron; PDK+V0120)--tubes weren't cloudy so we grew overnight cultures again
8 reactions (Ger S/A; BetS/A; Bet1.2 S/A; LTP S/A)
Annealing Temp: 65 C Extension Temp: 30 sec
Name
|
Amount (uL)
|
10mM dNTPs
|
1
|
5x Phusion Buffer
|
10
|
Phusion Polymerase
|
.5
|
DNA
|
1 uL
|
AB primer mix (1x)
|
2.5
|
Water
|
35
|
Concentrations: BetS: 109.1 ng/uL; BetA: 90.5 ng/uL; GerS: 69 ng/uL; GerA: 46 ng/uL; LTPS:36.7 ng/uL; LTPA:5.2 ng/uL; Bet1.2S: 77.4 ng/uL; Bet1.2A:66.2 ng/uL; PME: 64.2 ng/uL; PAL: 62.9 ng/uL; PDK: 35.7 ng/uL; PDK(2): 81.3 ng/uL; PDK(3): 21.5 ng/uL
|