IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/06/30

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6/30/10

Nanodropped PAL PCR cleanup—23.3 ng/ul

digestion

Changed digestion reaction since we only have ~400 ng DNA and protocol calls for 500 ng

DNA 20 ul (400 ng)
H2O 12.5 ul
Buffer 2 5 ul
BSA .5 ul
XbaI 1 ul
SpeI 1 ul

Incubate at 37º 15 min

Inactivate 80 º 20 min

Ligation:

H2O 13 ul
PAL digest 2 ul
pSB1A2 digest (from 6/29) 2 ul
Ligase Buffer 2 ul
Ligase 1 ul

RT 10 min

80ºC 20 min

Transformation:

100 ul competent cells + 2 ul PAL/pSB1A2 ligation. Electroporate, add 1mL LB, 1 hour recovery at 37ºC. No growth after 10 hours.

Colony Boil: MG1655

1 colony 2 colonies 3 colonies Swipe across lawn
20 ul H2O 20 ul H2O 20 ul H2O 20 ul H2O
96.0 ng/ul 96.0 ng/ul 96.0 ng/ul 96.0 ng/ul

Gradient PCR

ingredient amount/reaction 13 reactions (mastermix)
Primers 4 ul 52 ul
Template .69 ul (500 ng) 8.97 ul
Buffer 10X 4 ul 52 ul
10mM dNTPs 1 ul 13 ul
Polymerase .4 ul 5.2 ul
H2O 29.9 ul 388 ul

Each reaction gets 4 ul primers, 35.6 ul mastermix (which includes everything except primers). Ars and PAL primers at 10uM.

1. ArsB 45ºC

2. ArsB 48ºC

3. ArsB 51ºC

4. ArsB 54ºC

5. ArsB 57ºC

6. ArsB 60ºC

7. ArsB 63ºC

8. PAL

9. PAL (-) no primers

Program:

95°C 2 min
95°C 45 sec
45-63°C gradient 45 sec
72°C 90 sec
Go to step 2 33X
72°C 5 min
4°C hold

gel

lane sample amount
1. 1 Kb Ladder 2 ul
2. ArsB 45ºC 5 ul +1 ul dye
3. ArsB 48ºC 5 ul +1 ul dye
4. ArsB 51ºC 5 ul +1 ul dye
5. ArsB 54ºC 5 ul +1 ul dye
6. ArsB 57ºC 5 ul +1 ul dye
7. ArsB 60ºC 5 ul +1 ul dye
8. ArsB 63ºC 5 ul +1 ul dye
9. PAL 5 ul +1 ul dye
10. PAL (-) 5 ul +1 ul dye

1% TAE, 175V NO BANDS! Only ladder showed up!

digestions

J23100 J23119 Gas Vesicles Tet Backbone
DNA 2.17 ul 2.69 ul 1.16 ul 1.04 ul
H2O 40.33 ul 39.86 ul 41.34 ul 41.46 ul
Buffer 2 5ul 5ul 5ul 5ul
BSA .5 ul .5 ul .5 ul .5 ul
EcoRI 1 ul 1 ul 1 ul 1 ul
SpeI 1 ul 1 ul 1 ul 1 ul

37ºC incubation 15 min

80ºC inactivation 20 min

ligation

H2O 11 ul
J23100 2 ul
gas vesicles 2 ul
pSB1T3 2 ul
Ligase Buffer 2 ul
Ligase 1 ul

RT 10 min

80ºC 20 min

transformation

100 ul cells, 2 ul ligation (J23100-gas vesicles, J23119-gas vesicles) electroporate, add 1mL LB, 1 hour recovery at 37ºC. got no growth after 10 hours.

Gel

80V

Lane Sample Amount
1. 1 Kb ladder 2 ul
2. J23100 digestion 5 ul sample+ 1 ul dye
3. J23119 digestion 5 ul sample + 1 ul dye
4. Gas vesicle digestion 5 ul sample + 1 ul dye
5. pSB1T3 digestion 5 ul sample + 1 ul dye
6. J23100-Gas vesicles ligation 5 ul sample + 1 ul dye
7. J23119-Gas vesicles ligation 5 ul sample + 1 ul dye
8. pSB1T3 5 ul sample + 1 ul dye
9. PAL digest 5 ul sample + 1 ul dye
10. PAL ligation 5 ul sample + 1 ul dye

Ligation

H2O 13 ul
PAL digest (from this morning) 2 ul
pSB1A2 digest (from last night) 2 ul
Ligase Buffer 2 ul
Ligase 2 ul

incubated at room temp for 10 min, inactivated at 80°C for 20 min.

Warmed Amp/LB plate in 37°C room at 6:50 PM

7:15 PM

Transformation 100 ul competent cells and 2 ul ligation product in cuvette. electroporate and add 1 mL LB for recovery at 37°C 1 hour.

plated 20 ul on 1 half of Amp/LB plate, 200 ul on other half.



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