Team:KIT-Kyoto/Notebook/ATF2

ATF2

10th August

We performed PCR to amplify the ATF2 gene.

F:3' CGATCCCATGGGGATGGAAGATATAGAAGGATACGAACCACA

R:5' CGATCGGATCCAAAAGCGACGCAAATTCGCCGATGGTTTG

Buffer	50µL
dNTP	20µL
Primer mix	1µL
DNA sample	0.5µL
KOD-FX	2µL
H2O	26.5µL
Total	100µL

PCR products were electrophoresed in 1% agarose gel.

We detected a DNA band at around 1600bp.

PCR reactions were carried out (total volume was 300µL).

PCR products were electrophoresed in 1% agarose gel.

The PCR product with the size (1.6 kb) was verified as ATF2.

The PCR products of ATF2 was purified by ethanol precipitation

 

 

12th August

The PCR product of ATF2 were digested with the XhoI.

ATF2	5µL
Buff	2µL
H2O	12µL
Xho1	1µL

No DNA was detected.

We have repeated the PCR reactions to amplify ATF2 under the same conditions as those carried out on 10th, August.

 

 

 

14th August

1.

PCR reaction was carried out by using the following primer for pSB1C3.

F:3' CGATCTCTAGATGGAAGATATAGAAGGATACGAACCACAT

R:5' CGATCACTAGTATTAAAGCGACGCAAATTCGCCGATGGTTTG

PCR products were electrophoresed in 1% agarose gel.

The PCR product with the size (1.6 kb) was verified as ATF2.

The PCR products of ATF2 was purified by ethanol precipitation.

2.

The ATF2 gene and pET15b were purified by ethanol precipitation.

ATF2 was digested with the XhoI for verification.

ATF2	5µL
Buff	2µL
H2O	12µL
Xho1	1µL

PCR products were electrophoresed in 1% agarose gel.

The PCR products with the expected size were detected.

ATF2 treated ethanol precipitation and pET-15b was digested with the NcoI and BamHI.

pET-15b	87µL
Nco1	1µL
BamH1	1µL
Buff.	10µL
BSA	1µL
(total 100µL)

 

ATF2	24µL
Nco1	1µL
BamH1	1µL
Buff.	3µL
BSA	1µL
(total 30µL)

It was electrophoresed in 1% Blue gel.

ATF2 were extracted from the Blue gel.

 

17th August

pET-15b was digested with the Nco1 and BamH1.

pET-15b	87µL
Nco1	1µL
BamH1	1µL
Buff.	10µL
BSA	1µL
(total 100µL)

We treated the vector with 1µL BAP for 30 minutes.

It was electrophoresed in 1% Blue gel

ATF2 were extracted from the Blue gel

 

 

19th August

The ATF2 gene and pET-15b were purified.

We ligated pET-15b and ATF2.

ATF2	5µL
pET-15b	5µL
L.MIX	10µL
total 20µL

After the ligatation, we have transformed them into E.coli cells.

 

 

20th August

We got approximately 70 colonies and cultured them furthermore.

 

 

21th August

<ATF2 + pET15b>

We checked the insertion of DNA by colony cracking.

We could see correct bands of plasmid DNA only from five samples.

We cultured each transformant in LB liquid medium containing ampicillin.

PCR reaction was carried out using the following primers

1-1

F:3' TCGGCAATGCACAGTCATGGCCATAGTAGA

R:5' TTATTCAATTGGATGAGAAATCACCTTTATGAAATCATGCT

1-2

F:3' CATCTTGAATTCGATGACTTGATTATGAATAATCAACCA

R:5' CGATCACTAGTATTAAAGCGACGCAAATTCGCCGATGGTTTG

 

2-1

F:3' CGATCTCTAGATGGAAGATATAGAAGGATACGAACCACAT

R:5' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

 

2-2

F:3' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

R:5' ATCATCTTCGTAATCCTTGATGTGAATAGTGC

 

2.

PCR products were electrophoresed in 1% agarose gel.

The PCR products with the size (1.6 kb) were verified as ATF2.

The PCR products of ATF2 were purified by ethanol precipitation and digested with the following composition.

1-1

ATF2	25µL
Mfe1	2µL
Buff.	3µL
Total 30µL

 

1-2

ATF2	25µL
EcoR1	2µL
Buff.	3µL
Total 30µL

 

2-1

ATF2	25µL
Nsi1	2µL
Buff.	3µL
Total 30µL

 

2-2

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

 

It were electrophoresed in 1% Blue gel

ATF2 were extracted from the Blue gel

 

 

22th August

I

1

ATF2 gene extracted from the Blue gel was treated ethanol precipitation.

 

2

Ligation of 2-1 and 2-2 was performed by using Ligation mix.

1-1 with 1-2 and 2-1 with 2-2

The PCR products of ATF2 gene was purified.

It was electrophoresed in 1% agarose gel.

No DNA was detected.

 

II

1

PCR reaction was carried out using the primers.

The PCR products of ATF2 gene was purified.

It was digested with the following composition.

1-1

ATF2	25µL
Mfe1	2µL
Buff.	3µL
Total 30µL

 

1-2

ATF2	25µL
EcoR1	2µL
Buff.	3µL
Total 30µL

 

2-1

ATF2	25µL
Nsi1	2µL
Buff.	3µL
Total 30µL

 

2-2

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

It was electrophoresed in 1% Blue gel.

Any DNA fragment was not detected.

 

III

PCR reaction was carried out using the following primers.

1-1

F:3' TCGGCAATGCACAGTCATGGCCATAGTAGA

R:5' TTATTCAATTGGATGAGAAATCACCTTTATGAAATCATGCT

1-2

F:3' CATCTTGAATTCGATGACTTGATTATGAATAATCAACCA

R:5'

The PCR products of ATF2 gene was purified.

It was digested with the following composition.

1-1

ATF2	25µL
Mfe1	2µL
Buff.	3µL
Total 30µL

 

1-2

ATF2	25µL
EcoR1	2µL
Buff.	3µL
Total 30µL

It were electrophoresed in 1% Blue gel.

ATF2 were extracted from the Blue gel.

ATF2 extracted from the Blue gel was purified.

 

Ⅳ

PCR reaction was carried out using the primer.

The PCR products of ATF2 gene was purified.

It was digested with the following composition.

1-1

ATF2	25µL
Mfe1	2µL
Buff.	3µL
Total 30µL

 

1-2

ATF2	25µL
EcoR1	2µL
Buff.	3µL
Total 30µL

 

 

23th August

The plasmids (d, e, f, I, o) were digested with Nco1 and BamH1.

We applied them to 1% agarose gel electrophoresis and checked them.

Sample d, e, f, i had two bands and we cultured them.

The plasmids (d, e, f, I,) were digested with BsiWI and checked.

We detected the correct size band.

We minipreped and transformed them into BL21(DE3).

 

 

 

I

Sample 1.

ATF2 gene (Ⅳ) extracted from the Blue gel was purified by ethanol precipitation.

Sample  2.

ATF2 gene (Ⅲ) extracted from the Blue gel was purified by ethanol precipitation.

Ligation of sample 1 and 2 was performed by Ligation mix.

PCR reaction was carried out using the primers

PCR products were electrophoresed in 1% agarose gel, and several fragments were detected.

 

II

PCR reaction was carried out using the primers

1-1

F:3' TCGGCAATGCACAGTCATGGCCATAGTAGA

R:5' TTATTCAATTGGATGAGAAATCACCTTTATGAAATCATGCT

1-2

F:3' CATCTTGAATTCGATGACTTGATTATGAATAATCAACCA

R:5' CGATCACTAGTATTAAAGCGACGCAAATTCGCCGATGGTTTG

The PCR products of ATF2 gene were purified, and digested with Mfe and EcoRⅠ.

After the digestion, the ATF2 gene was electrophoresed in 1% Blue gel.

The ATF2 genes were extracted from the Blue gel and purified.

Ligation of 2-1 and 2-2 was performed using Ligation mix.

PCR reaction was carried out using the primers

F:3' CGATCTCTAGATGGAAGATATAGAAGGATACGAACCACAT

R:5' CGATCACTAGTATTAAAGCGACGCAAATTCGCCGATGGTTTG

 

 

24th August

I

PCR reaction was carried out using the following primers.

2-1

F:3' CGATCTCTAGATGGAAGATATAGAAGGATACGAACCACAT

R:5' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

 

2-2

F:3' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

R:5' ATCATCTTCGTAATCCTTGATGTGAATAGTGC

 

The PCR products of ATF2 gene was purified.

It was digested with the following composition.

2-1

ATF2	25µL
Nsi1	2µL
Buff.	3µL
Total 30µL

 

2-2

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

It was electrophoresed in 1% Blue gel.

No DNA was detected.

 

II

１

PCR react ion was carried out using the primer.

The PCR products for ATF2 was treated ethanol precipitation.

It was digested with the following composition.

2-1

ATF2	25µL
Nsi1	2µL
Buff.	3µL
Total 30µL

 

2-2

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

It were electrophoresed in 1% Blue gel.

ATF2 were extracted from the Blue gel.

ATF2 extracted from the Blue gel was purified.

Ligation of 2-1 and 2-2 was performed using Ligation mix.

 

III

1.

PCR reaction was carried out using the primer.

The PCR products of ATF2 gene was purified.

It was digested with the following composition.

ATF2	25µL
EcoR1	2µL
Buff.	3µL
Total 30µL

It was electrophoresed in 1% agarose gel.

Multiple DNA fragments were detected.

 

IV

１

PCR reaction was carried out using the primers.

The PCR products of ATF2 gene was purified.

It was digested with the following composition.

ATF2	25µL
EcoR1	2µL
Buff.	3µL
Total 30µL

It was electrophoresed in 1% agarose gel.

Multiple DNA fragments were detected for the DIAP2.

 

26th August

Cell free extract was prepared and applied to SDS-PAGE to detect ATF2 protein.

 

 

27th August

PCR products were electrophoresed in 1% agarose gel.

The PCR products were treated ethanol precipitation.

They were digested.

They were electrophoresed in 1% Blue gel.

ATF2 were extracted from the Blue gel.

ATF2 extracted from the Blue gel was treated ethanol precipitation.

Ligation of 2-1 and 2-2 was performed using Ligation mix.

We purified them.

 

28th August

The PCR products was treated ethanol precipitation.

PCR reaction was carried out by using the primer.

PCR products were electrophoresed in 1% agarose gel.

No DNA was detected.

We performed PCR, purified, and digested again.

We ligated and purified them.

 

 

29th August

I

PCR reaction was carried out using the primer.

PCR products were electrophoresed in 1% agarose gel.

No DNA band was detected.

 

II

PCR reaction was carried out using the following primers.

2-1

F:3' CGATCTCTAGATGGAAGATATAGAAGGATACGAACCACAT

R:5' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

 

2-2

F:3' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

R:5' ATCATCTTCGTAATCCTTGATGTGAATAGTGC

 

The PCR products were treated ethanol precipitation.

It was digested with the following composition.

2-1

ATF2	25µL
Nsi1	2µL
Buff.	3µL
Total 30µL

 

2-2

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

It was electrophoresed in 1% Blue gel.

ATF2 were extracted from the Blue gel.

ATF2 extracted from the Blue gel was purified.

We ligated them and purified it.

We performed PCR to amplify it.

 

 

Cell free extract was prepared with FastBreak Cell Lysis Reagent and applied to SDS-PAGE.

 

30th August

I

1

PCR reaction was carried out using the following primers

1-1

F:3' TCGGCAATGCACAGTCATGGCCATAGTAGA

R:5' TTATTCAATTGGATGAGAAATCACCTTTATGAAATCATGCT

1-2

F:3' CATCTTGAATTCGATGACTTGATTATGAATAATCAACCA

R:5'

The PCR products were purfied.

It was digested with the following composition.

2-1

ATF2	25µL
Nsi1	2µL
Buff.	3µL
Total 30µL

 

2-2

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

It were electrophoresed in 1% Blue gel.

ATF2 were extracted from the Blue gel.

ATF2 extracted from the Blue gel was purified.

We ligated them and purified it.

PCR reaction was carried out using the following primers for pSB1C3.

PCR products were electrophoresed in 1% agarose gel.

The PCR products of ATF2 gene was purified.

It was digested with the following composition.

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

PCR products were electrophoresed in 1% agarose gel.

The PCR products of ATF2 gene was purified.

 

II

１

We performed PCR (prepared 8/29).

PCR reaction was carried out using the following primers.

2-1

F:3' CGATCTCTAGATGGAAGATATAGAAGGATACGAACCACAT

R:5' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

 

2-2

F:3' CGCAGTCTGCAGGCGCCTTGAGATTTGCGTTCGGCCTAA

R:5' ATCATCTTCGTAATCCTTGATGTGAATAGTGC

 

The PCR products were purified.

It was digested with the following composition.

2-1

ATF2	25µL
Nsi1	2µL
Buff.	3µL
Total 30µL

 

2-2

ATF2	25µL
Pst1	2µL
Buff.	3µL
Total 30µL

It was electrophoresed in 1% Blue gel.

ATF2 was extracted from the Blue gel.

ATF2 extracted from the Blue gel was purified.

We ligated it.

We purified it and performed PCR.

 

 

2^nd September

We purified the mutant of ATF2.

We digested it and cc3 with Xba1 and Spe1.

We electrophoresed and extracted them in and from 1% Blue gel.

We purified them.

We ligated and transformed them into E.coli cells.

 

 

4^th September

We purified the mutant of ATF2.

Three tubes of pSB1C3 were treated ethanol precipitation

We add 25mL H₂O and digested it with Xba1 and Spe1.

We electrophoresed and extracted them in and from 1% Blue gel.

We purified them.

We ligated and transformed them into E.coli cells.

 

 

5^th September

We did rapid check of the insert by colony cracking (ATF2+pSB1C3).

But we detected no band.

We cultured them for 5 hours and minipreped.

We checked them by electrophoresing but couldn’t see any bands.

So, we thought them failed.

We cultured other sample (ATF2+pSB1C3).