IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/08

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7/8/10

11:00 AM

Colony Boil

25 ul H2O, swiped across plate to pick up a bunch of colonies. incubate at 95°C for 2 min. nanodropped, 444.6 ng/ul.

PCR

Ingredient Amount per reaction Mastermix (9 reactions)
10X buffer 4 ul 36 ul
10 mM dNTPs 1 ul 9 ul
Template 1.12 ul (500ng) 10.08 ul
F Primer 4 ul added individually to each reaction
R Primer 4 ul added individually to each reaction
Polymerase .4 ul 3.6 ul
H2O 25.48 ul 229.32 ul

Per Reaction: 32 ul Mastermix, 4 ul F Primer, 4 ul R Primer

Samples:

pArsR F1R1(540 bp)

pArsR F1R2 (547 bp)

pArsR F2R1 (293 bp)

pArsR F2R2 (300 bp)

ArsR 1

ArsR 2

negative control (no primers)

Primers (10mM):

pArsR F1

pArsR F2

pArsR R1

pArsR R2

ArsR F RFC10 EcoRI

ArsR R RFC10

Program:

95°C 2 min
95°C 30 sec
55°C 30 sec
72°C 1 min
go to step 2 31X
72°C 5 min
hold at 4°C

2:05 PM

gel

Lane Sample Amount
1. 1 Kb Ladder 1 ul
2. pArsR F1R1 5ul sample, 1 ul dye
3. pArsR F1R2 5ul sample, 1 ul dye
4. pArsR F2R1 5ul sample, 1 ul dye
5. pArsR F2R2 5ul sample, 1 ul dye
6. ArsR1 5ul sample, 1 ul dye
7. ArsR2 5ul sample, 1 ul dye
8. negative control 5ul sample, 1 ul dye

90V

Blank gel again

2:30 PM

PCR #a million of LamB and ArsR and pArsR - NEW MIXTURE

Ingredient Amount per 20ul reaction Mastermix (9 reactions)
10X buffer 2 ul 18 ul
10 mM dNTPs .4 ul 3.6 ul
Template 1 ul (20ng) 9 ul
10uM F Primer .4 ul added individually to each reaction
10uM R Primer .4 ul added individually to each reaction
Pfu Turbo DNA Polymerase .2 ul 31.8 ul
H2O 15.6 ul 140.4 ul

Per Reaction: 32 ul Mastermix, 4 ul F Primer, 4 ul R Primer

Samples:
1. LamB F1R
2. LamB F2R
3. ArsR
4. pArsR F1R1(540 bp)
5. pArsR F1R2 (547 bp)
6. pArsR F2R1 (293 bp)
7. pArsR F2R2 (300 bp)
8. negative control (no primers)

Program:

95°C 3 min
95°C 30 sec
55°C 30 sec
72°C 90 sec
go to step 2 31X
72°C 5 min
hold at 4°C
  • The gel for this PCR was run on Monday July 12th


3:25

digestions

pArsR

DNA 10 ul (500 ng)
H2O 32.5 ul
Buffer 2 5 ul
BSA .5 ul
SpeI 1 ul
XbaI 1 ul

pSB1T3

DNA 1 ul (500 ng)
H2O 41.5 ul
Buffer 2 5 ul
BSA .5 ul
SpeI 1 ul
XbaI 1 ul

Incubate at 37°C for 15 min, inactivate at 80°C for 20 min

PCR purified pSB1T3 and SAP: 2 ul SAP buffer, 1 ul SAP 37°C 10 min, 65°C 15 min.

4:45

PCR

Ingredient Amount per reaction Mastermix (9 reactions)
10X Buffer 4 ul 36 ul
10 mM dNTPs 1 ul 9 ul
Template 1.12 ul (500 ng) 10.08 ul
F Primer 4 ul added individually to each reaction
R Primer 4 ul added individually to each reaction
Pol .4 ul 3.6 ul
H2O 25.48 ul 229.32 ul

Per Reaction: 32 ul mastermix, 4 ul F primer, 4 ul R primer

Primers:

pArsRF1

pArsRF2

pArsRR1

pArsRR2

ArsR F RFC10 EcoRI

ArsR R RFC10

Samples:

pArsR F1R1 540bp

pArsR F1R2 547bp

pArsR F2R1 293 bp

pArsR F2R2 300 bp

ArsR

Negative control (no primers

Program

95°C 5 min
95°C 30 sec
55°C 30 sec
72°C 1 min
go to step 2 31X
72°C 5 min
hold at 4°C

SAP Backbone

2.6 ul SAP buffer

1.3 ul SAP

Incubate at 37°C for 10 min, inactivate at 65°C for 15 min.

Gel

Lane Sample Amount
1 1 Kb Ladder 1 ul
2 Empty N/A
3 Empty N/A
4 pArsR (7/8/10) 50 ul sample, 10 ul dye
5 Empty N/A
6 pSB1T3/SAP (7/8/10) 30 ul sample, 6 ul dye
Lane Sample Amount
1 pSB1T3/SAP (7/1/10) 50 ul sample, 10 ul dye
2 Empty N/A
3 pArsR (7/7/10) 50 ul sample, 10 ul dye
4 Empty N/A
5 Empty N/A
6 1 Kb Ladder 1 ul

8:40 PM

Band Extraction Zymogen Kit

pSB1T3 (7/1) and pArsR (7/7). the other samples didn't show up on gel.

Concentrations post-band extraction:

pSB1T3 8 ng/ul

pArsR 20 ng/ul

9:20 PM

Ligations

H2O 13 ul
pSB1T3 2 ul
pArsR 2 ul
10X Ligase Buffer 2 ul
Ligase 1 ul

Incubate at 14°C overnight, inactivate at 80°C for 20 min tomorrow.