Team:KIT-Kyoto/Notebook/ATF1/august

From 2013.igem.org

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<h2>ATF1</h2>
<h2>ATF1</h2>
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<p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>August 6th</span></b></p>
<p class=MsoNormal><b><span lang=EN-US style='font-size:14.0pt'>August 6th</span></b></p>
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<p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Digested pET-15b
<p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Digested pET-15b
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with XhoI at 37</span><span style='font-size:11.0pt;font-family:メイリオ'>&#730;<span
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with <i>Xho</i>I at 37</span><span style='font-size:11.0pt;font-family:メイリオ'>&#730;<span
lang=EN-US>C</span></span><span lang=EN-US style='font-size:11.0pt'> for 3
lang=EN-US>C</span></span><span lang=EN-US style='font-size:11.0pt'> for 3
hours.</span></p>
hours.</span></p>
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<p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Digested pET-15b
<p class=MsoNormal><span lang=EN-US style='font-size:11.0pt'>Digested pET-15b
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with Bpu1102I at 37</span><span style='font-size:11.0pt;font-family:メイリオ'>&#730;<span
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with <i>Bpu</i>1102I at 37</span><span style='font-size:11.0pt;font-family:
-
lang=EN-US>C</span></span><span lang=EN-US style='font-size:11.0pt'> for 3
+
メイリオ'>&#730;<span lang=EN-US>C</span></span><span lang=EN-US style='font-size:11.0pt'>
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hours.</span></p>
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for 3 hours.</span></p>
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0

Latest revision as of 09:18, 27 September 2013



KIT-Kyoto

ATF1

August 6th

The ATF1 gene was purified (prepared on 7/19)

Digested pET-15b with XhoI at 37 ˚C for 90 minutes.

pET-15b

88µL

XhoI Buffer

10µL

XhoI

2µL

Total

100µL

Purified pET-15b DNA was dissolved in 34µL of H2O.

Digested pET-15b with Bpu1102I at 37 ˚C for 90 minutes.

pET-15b

34µL

Bpu1102I Buffer

4µL

Bpu1102I

2µL

Total

40µL

Added 1uL of BAP to the solution and incubated it at 37 ˚C for 30 minutes.

Applied ATF1 and pET-15b to the blue gel electrophoresis.

Results: No band was detected.

Cultivated pET-15b in 3mL LB medium with ampicillin overnight.

 

August 7th

Minipreped pET-15b DNA.

Digested pET-15b with XhoI at 37˚C for 3 hours.

pET-15b

88µL

XhoI Buffer

10µL

XhoI

2µL

Total

100µL

Purified it and dissolved in 26µL of H2O.

Digested pET-15b with Bpu1102I at 37˚C for 3 hours.

pET-15b

26µL

Bpu1102I Buffer

3µL

Bpu1102I

1µL

Total

30µL

Added 1µL of BAP to this solution and incubated it at 37˚C for 30 minutes.

Applied ATF1 and pET-15b to the blue gel electrophoresis.

 

August 8th

Digested pET-15b with XhoI at 37˚C overnight.

pET-15b

89µL

XhoI Buffer

10µL

XhoI

1µL

Total

100µL

 

 

August 9th

Purified pET-15b (prepared on 8/8) and added 26ul of H2O.

Digested pET-15b with Bpu1102I at 37˚C for 3 hours.

pET-15b

26

Bpu1102I Buffer

3µL

Bpu1102I

1µL

Total

30µL

Added 1µL of BAP to this solution and incubate it at 37˚C for 30 minutes.

Applied pET-15b to the blue gel electrophoresis.

Isolated and Purified it.

Ligation pET-15b and ATF1 (prepared 7/19,8/6) at room temperature for 15 minutes.

ATF1

5µL

pET-15b

5µL

Ligation MIX

10µL

Total

20µL

The ATF1 into pET-15b was transformed into E.coli cells.

Cultivated transformants on LB plate with ampicillin at37˚C overnight.

 

August 10th

Picked 48 colonies of ATF1 transformants.

 

 

August 12th

Checked the colonies by colony cracking.

Picked up the appropriate colonies and cultured in the LB in 3mL ampicillin(+) medium at 37˚C for 4 hours.

Miniprepped plasmid DNA.

 

 

August 13th

Digested plasmid DNA (prepared on 8/12) with HindIII at 37˚C for 90 minutes.

Plasmid DNA

5µL

Buffer

2µL

H2O

12µL

HindIII

1µL

Total

20µL

Applied pET-15b to the agarose gel electrophoresis.

Digested plasmid DNA (prepared on 8/12) with HindIII at 37˚C for 90 minutes.

Plasmid DNA

10µL

Buffer

2µL

H2O

7.25µL

HindIII

0.75µL

Total

20µL

Applied pET-15b to the agarose gel electrophoresis.

Again, picked up the appropriate colonies and cultured in the 3mL LB medium with ampicillin at 37˚C.

 

 

 

August 26th

We performed PCR to amplify the ATF1 gene.

Buffer

50µL

dNTP

20µL

Primer mix

1µL

Genome DNA

0.5µL

KOD-FX

2µL

H2O

26.5µL

 

 

August 27th

Purified PCR products (prepared on 8/26) and added 30µL of H2O.

Digested ATF1 with HindIII.

ATF1

5µL

Buffer

2µL

HindIII

2µL

H2O

11µL

Digested pET-15b and ATF1 with XhoI.

ATF1

25µL

Buffer

3µL

XhoI

2µL

 

pET-15b

100µL

Buffer

11.3µL

XhoI

2µL

Purified it and added 25µL of H2O.

Digested pET-15b and ATF1 with Bpu1102I.

ATF1

25µL

Buffer

3µL

Bpu1102I

2µL

 

pET-15b

25µL

Buffer

3µL

Bpu1102I

2µL

Applied pET-15b and ATF1 to the blue gel electrophoresis.

Purified PCR products.

 

 

August 28th

Digested ATF1 (prepared on 8/27) with EcoRI.

ATF1

5µL

Buffer

1µL

EcoRI

2µL

H2O

2µL

Applied it to the agarose gel electrophoresis.

Digested ATF1 (prepared on 8/27) and pET-15b with XhoI.

ATF1

25µL

Buffer

3µL

XhoI

2µL

 

pET-15b

100µL

Buffer

11.3µL

XhoI

2µL

 

 

August 29th

Applied ATF1 and pET-15b to the blue gel electrophoresis.

Ligated it.

ATF1

10µL

pET-15b

10µL

Ligation  MIX

10µL

Carried out transformation.

 

 

August 30th

Checked the colonies by colony cracking.

We performed PCR to amplify the ATF1 gene.

Buffer

50µL

dNTP

20µL

Primer mix

1µL

Genome DNA

0.5µL

KOD-FX

2µL

H2O

26.5µL

 

 

 

August 31st

Purified PCR products and dissolved 30µL of H2O.

Purified pET-15b and added 33µL of H2O.

Digested ATF1 and pET-15b with XhoI and Bpu1102I.

NEB Buffer 2

3µL

ATF1

24µL

BSA

1µL

XhoI

1µL

Bpu1102I

1µL

 

NEB Buffer 2

4µL

pET-15b

33µL

BSA

1µL

XhoI

1µL

Bpu1102I

1µL

Added 1µL of BAP to pET-15b and incubated them at 37˚C for 30 minutes.

Applied pET-15b to the blue gel electrophoresis.

Isolated and purified it.

Ligation of pET-15b with ATF1 (prep).

ATF1

10µL

pET-15b

10µL

Ligation  MIX

10µL

Transformed ATF1 into pET-15b E. coli cells.

 

 

 

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