Team:Biwako Nagahama/Material & Method

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Contents

Material & Method

CelC

Agro Notebook

5/31 Cloning of CelC and Restriction Enzyme

By Koki Tsutsumi

CelC gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CelC gene has restriction enzyme sites,EcoRI and BamHI.

Biwako-Nagahama T.Ksenpai celC1.png

Inverse PCR

Biwako-Nagahama T.Ksenpai celC1.png

※Restriction Enzyme sol.(line No.5 CelC_EcoRI)which has 1M NaCl dissolved in it, so, the band in lane no.5 appeared as upper-side.

CelC_Fw: TGACGAAAGCACTGATCTGC

CelC_Rv: GAAAAGATCGAAACGGTGG

Biwako-Nagahama T.Ksenpai celC3.png

TA cloning of CelC

Biwako-Nagahama T.Ksenpai celC4.png

16℃ 30min incubate

Ligation of CelC/pMD20 and Transformation in JM109.

Biwako-Nagahama T.Ksenpai celC5.png


Biwako-Nagahama T.Ksenpai celC6.png

Cells were stored on ice for 30min.

After 42℃ 30sec heat shock, cells were stored on ice for 2min.

Then cells were pre-cultured at 37℃ for 1hr, plated to Ampicillin plate.

6/1 Liquid clluture

CelC/pMD20 22 samples at 37°C, for overnight.

6/2 MiniPrep of CelC

Biwako-Nagahama T.Ksenpai celC7.png

Biwako-Nagahama T.Ksenpai celC8.png

Linear CelC/pMD20 DNA :2736bp. This CelC/pMD20 sample is cccDNA. So,white 6,white 7,white 8,white 9,white 14 probably picked up CelC/pMD20.

6/3 Restriction Enzyme of CelC/pMD20

CelC gene had produced clone from Agrobacterium tumefaciens C58, I confirmed whether it’s true or not. CelC/pMD20 gene has 2 restriction enzyme sites,BamHI

Biwako-Nagahama T.Ksenpai celC9.png


Biwako-Nagahama T.Ksenpai celC10.png

I confirmed the direction of CelC gene.

6/ Sequence of CelC/pMD20

Biwako-Nagahama T.Ksenpai celC11.png

7/18 PointMutation of CelC

CelC gene had Restriction Enzyme Site,EcoRI. We directed the EcoRI site.

Biwako-Nagahama T.Ksenpai celC12.png

Biwako-Nagahama T.Ksenpai celC13.png


Front

Fw1 Primer:

TATATATTCTAGATGAAGAGCGGGATTTCG

Rv2 Primer:

CATTATATCCGAACTCCGGCTG

Rear

Fw2 Primer:

AGCCGGAGTTCGGATATAATGC


Rv1 Primer:

CAGCACGAACTAGTATTATTATCATCGGC

Biwako-Nagahama T.Ksenpai celC14.png


7/19 Gel Purification of CelC gene’s Front Fragment and Rear Fragment

Front Fragment DNA and Rear Fragment DNA that 765bp and 347bp band performed Gel Purification by illustra GFX PCR Purification Kit.

7/21 CelC gene’s Front Fragment and Rear Fragment Overlap PCR

No.6,7,8,9,14 each fragment Overlap PCR completed.

I selected No.8.

7/22 Gel Purification of CelC gene No.8

No.8 DNA that about 1ooo bp band performed Gel Purification by illustra GFX PCR Purification Kit.

8/1 Adapter PCR of CelC gene No.8 (BioBrick Part)

Fw Primer:

GTTTCTTCGAATTCGCGGCCGCTTCTAGATG

Rv Primer:

GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATC

8/11 BioBrick of CelC Restrict Enzyme ,EcoRI.

I confirmed BioBrick of CelC non-Restriction Enzyme ,EcoRI.

9/5 Brick Part of CelC and pSB1C3 Restrict Enzyme,EcoRI and PstI.

9/5 Ligation of CelC/pSB1C3 and Transformation in JM109.

9/7 Colony PCR of CelC/pSB1C3

CelC/pSB1C3 DNA(VR-VF2) :1370bp. So,No.5 and No.12 probably picked up CelC/pSB1C3

9/8 Miniprep of CelC/pSB1C3

Linear CelC/pSB1C3 DNA :3126bp. This CelC/pSB1C3 sample is cccDNA. So,No.5 and No.12 probably picked up CelC/pSB1C3.

9/15. Sequence of CelC Brick Part sequence.

Result of NCBI BLAST


CrdS

Cloning of CrdS and Restriction Enzyme

By Koki Tsutsumi

CrdS gene had produced clone from Agrobacterium tumefaciens C58, but I confirmed whether it’s true or not. CrdS gene has restriction enzyme sites,EcoRI and PstI.

Biwako-Nagahama E.P tutumi1.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS12μL
4--
5--
6--
7--
8--
Biwako-Nagahama E.P tutumi2.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS_nonRE12μL
4CrdS/EcoRⅠ12μL
5CrdS/PstⅠ12μL
6λ-HindⅢ10μL
7500bp DNA ladder10μL
8--

Inverse PCR

Prime STAR MAX25μL
10pmol/μL Primer F1μL
10pmol/μL Primer R1μL
PCR反応液(2ng)1μL
dH2O22μL
Total22μL

CrdS_Fw: AGTACGATCCGCTATTTTCCCG

CrdS_Rv: CAGACCAAGATTTCGCGAACTC


94℃ 2min

98℃ 10sec

48℃ 30sec

72℃ 2min

72℃ 2min


TA cloning of CrdS

PCR product1μL
pMD202μL
Ligation kit ver.23μL

16℃ 30min incubate

Ligation of CrdS/pMD20 and Transformation in JM109

Competent cell100μL
DNA6μL

↓Cells were stored on ice for 30min.

↓After 42℃ 30sec heat shock, cells were stored on ice for 2min.

↓Then cells were pre-cultured at 37℃ for 1hr, plated to Ampicillin plate.

Liquid culluture

CrdS/pMD20 21 samples at 37℃,for overnight

MiniPrep of CrdS/pMD20

Linear CrdS/pMD20 DNA :4995bp. This CrdS/pMD20 sample is cccDNA. So, probably picked up CrdS/pMD20.

Biwako-Nagahama E.P tutumi3.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS No.12μL
4CrdS No.12μL
5CrdS No.12μL
6CrdS No.12μL
7CrdS No.12μL
8CrdS No.12μL

Restriction Enzyme of CrdS/pMD20

CrdS gene had produced clone from Agrobacterium tumefaciens C58, I confirmed whether it’s true or not. CrdS/pMD20 gene has 2 restriction enzyme sites,EcoRI.

Biwako-Nagahama E.P tutumi4.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS No.12μL
4CrdS No.12μL
5CrdS No.12μL
6CrdS No.12μL
7CrdS No.12μL
8CrdS No.12μL

I comfirmed the direction of CrdS gene.

Sequence of CrdS/pMD20

Biwako-Nagahama Sequences tutumi5.png

PointMutation of CrdS

CrdS gene had Restriction Enzyme Site,EcoRI. We removed the EcoRI site.

Biwako-Nagahama E.P tutumi6.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS Negative Front Fragment12μL
4CrdS Negative Rear Fragment12μL
5CrdS No.6 Front Fragment12μL
6CrdS No.6 Middle Fragment12μL
7CrdS No.6 Rear Fragment12μL
8CrdS No.15 Front Fragment12μL
9CrdS No.15 Middle Fragment12μL
10CrdS No.15 Rear Fragment12μL
11--
12--
13--
14--
15λ-HindⅢ10μL
16500bp DNA ladder10μL

Front

Fw0 Primer:GGCCGCTTCTAGATGTATTTCAGTGC

Rv1 Primer:CGTTTCGAGGGAGAACTCCAGCG

Middle

Fw1Primer:CGCTGGAGTTCTCCCTCGAAACG

Rv2Primer:CAGCTCATCGGCCTGAAGCGCC

Rear

Fw2 Primer:GGCGCTTCAGGCCGATGAGCTG

Rv0 Primer:GGCGCTACTAGTATTATTATCACCCGAATG

5xPS Buffer10μL
dNTP Mixture4μL
10pmol/µl Primer Fw1 or Fw21μL
10pmol/µl Primer Rv2 or Rv11μL
Templete1μL
Prime STAR HS0.5μL
dH2O32.5μL
Total50μL

94℃ 2min

98℃ 10sec

55℃ 5sec

72℃ 70sec

4℃ ∞


Gel Purification of CrdS gene’s Front Fragment and Rear Fragment

Front Fragment DNA and Rear Fragment DNA ,Middle Fragment DNA that 223 bp and 1037 bp ,782 bp band performed Gel Purification by illustra GFX PCR Purification Kit.

CrdS gene’s Front Fragment and Rear Fragment Overlap PCR

Biwako-Nagahama E.P tutumi7.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS N Front+Middle DNA12μL
4CrdS No.6 Front+Middle DNA12μL
5CrdS No.15 Front+Middle DNA12μL
6λ-HindⅢ10μL
7500bp DNA ladder10μL
8--
Biwako-Nagahama E.P tutumi8.png

NoNamevolume
1500bp DNA ladder10μL
2λ-HindⅢ10μL
3CrdS N Front+Rear DNA12μL
4CrdS No.6 Front+Rear DNA12μL
5CrdS No.15 Front+Rear DNA12μL
6λ-HindⅢ10μL
7500bp DNA ladder10μL
8--

N, No.6, No.15 each fragment Overlap PCR completed.

Gel Purification of CrdS

No.8 DNA that about 2ooo bp band performed Gel Purification by illustra GFX PCR Purification Kit.

Adapter PCR of CrdS gene (BioBrick Part)

Biwako-Nagahama E.P tutumi9.png

NoNamevolume
12-log DNA ladder5μL
2λ-HindⅢ6μL
3PCR saample18μL
4--
5--
6--
7--
8--

Fw Primer: GTTTCTTCGAATTCGCGGCCGCTTCTAGATG Rv Primer: GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATC

Brick Part of CrdS Restrict Enzyme,EcoRI and PstI.

Biwako-Nagahama E.P tutumi10.png