Team:KIT-Kyoto/Notebook-week2p

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Revision as of 15:18, 25 September 2012

August 31st

1. PCR amplification of DNA fragments containing UAS and Heat Shock promoter
We have carried out the PCR reaction for UAS again. PCR amplification of Heat Shock promoter from pCasPer DNA was also carried out in the following reactions.

DNA template	0.5uL
10× KOD plus buffer	10uL
2mM dNTPs	10uL
25mM MgSO₄	3.2uL
10P 5’ primer	3uL
10P 3’ primer	3uL
KOD plus	2uL
dH₂O	68.3uL
Total	100uL

Reaction conditions

Temperature	Time	Cycle
95℃	2min
95℃	15sec	25 cycle
58℃	30sec	25 cycle
68℃	30sec	25 cycle
68℃	30sec
14℃	∞

2. Purification of pAct5C DNA and pGaTB DNA
pAct5C DNA and pGaTB DNA was purified from E. coli prepared on 8/29by QIA prep Spin Miniprep Kit.

September 1st

1. Agarose gel electrophoresis of PCR produsts and the purified pAct5C DNA and pGaTB DNA
Agarose gel image is shown below. Left to right: HS promoter 10uL, UAS fragment 10uL, pAct5C DNA-1, pGaTB DNA-1, pGaTB DNA-2, pAct5C DNA

2. Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.

September 3rd

1. PCR amplification of DNA fragments containing GAL4, Act5C promoter and LacZ sequence
pGaTBDNA was used as a template to amplify GAL4 sequence. pAct5C-LacZ DNA was used to amplify Act5C promoter-enhancer region and LacZ.

DNA template	0.2uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO₄	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1u　L
dH₂O	34.2uL
Total	50uL

Reaction conditions

Temperature	Time	Circle
95℃	2min
95℃	15sec	25 cycle
55℃(GAL4) and 58℃(Except for GAL4)	30sec	25 cycle
68℃	2min30sec	25 cycle
68℃	2min30sec
14℃	∞

2. Agarose gel electrophoresis of the PCRproducts
Left to right: 1kb ladder marker 5uL　GAL4 fragments 10uL　Act5C promoter-enhancer 10uL　LacZ sequence

Results: Expected PCR products were all detected on the gel.
These PCR products were purified by High Pure PCR Product Kit (Roche).

September 4th

1. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ

GAL4 fragments prepared on 9/3	40uL
M buffer(TOYOBO)	5uL
XbaⅠ(TOYOBO)	0.5uL
SpeⅠ(TOYOBO)	0.5uL
dH₂O	4uL
Total	50uL

2. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.

3. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ
pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.

each DNA	40uL
3 buffer(NEB)	5uL
BglⅡ(NEB)	0.5uL
dH₂O	4.5uL
Total	50uL

Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.

pSB1C3 DNA	23uL
M buffer(TOYOBO)	10uL
XbaⅠ(TOYOBO)	1uL
SpeⅠ(TOYOBO)	1uL
CIP	0.5uL
dH₂O	64.5uL
Total	100uL

4. Purification of GAL4 fragments
After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit

Photo of agarose gel

5. Purification of BglII-digested LacZ fragments
BglII-digested LacZ fragments were purified by High Pure PCR Product Kit.

September 5th

1. Purification of EGFP fragments and HS promoter fragments
EGFP fragments prepared on 8/29 and HS promoter fragments prepared on 8/31were purified by High Pure PCR Product Kit

2. EGFP fragments and pSB1C3 DNA prepared on 8/27were digested with BglⅡ in the following reaction.

	EGFP	pSB1C3
DNA template	40uL	23uL
3 buffer	5uL	5uL
BglⅡ	0.5uL	0.5uL
dH₂O	4.5uL	21.5uL
Total	50uL	50uL

3. Purification of pSB1C3DNA digested with XbaⅠ and SpeⅠ
Agarose gel electrophoresis image of the purified sample are shown below.

4. SpeI digestion of the BglⅡ-digested LacZ, EGFP, pSB1C3 DNA
SpeⅠ digestion was carried out in the following reactions.

DNA template	40uL
4 buffer(NEB)	5uL
SpeⅠ	0.5uL
100×BSA	0.5uL
CIP	0.5uL
dH₂O	4uL
Total	50uL

5. Ligation of pSB1C3 DNA and GAL4 fragment
Ligation was carried out in the following reactions.

pSB1C3 (XbaⅠand SpeⅠdigested)	1uL
GAL4 (XbaⅠ and SpeⅠ digested)	1uL
dH₂O	3uL
Ligation high	2.5uL
Total	7.5uL

6. The ligated products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(＋) plate

7. Purification of pSB1C3, EGFP, LacZfragments double digested with BglⅡ and SpeⅠ
The double digested samples were applied to the agarose gel electrophoresis and the DNA fragments were purified by QIA quick Gel Extraction Kit

Photo of agarose gel

>>>>>>>>>WEEK3

@@ Line 149: / Line 149: @@
 <br><br>
 <img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300">
-<br><br>
-<Table Border Cellspacing="0">
-<Tr><Td></Td><Td>　All samples　</Td></Tr>
-<tr><td>　DNA template　</td><td>　0.3uL　</td></tr>
-<Tr><Td>　10× KOD plus buffer　</Td><Td>　10uL　</Td></Tr>
-<tr><td>　2mM dNTPs　</td><td>　10uL　</td></tr><tr>
-<td>　25mM MgSO<sub>4</sub>　</td><td>　3.2uL　</td></tr>
-<td>　10P 5’ primer　</td><td>　3uL　</td></tr>
-<td>　10P 3’ primer　</td><td>　3uL　</td></tr>
-<td>　KOD plus　</td><td>　2uL　</td></tr>
-<td>　dH<sub>2</sub>O　</td><td>　68.5uL　</td></tr>
-<td>　Total　<td>　100uL　</td></tr>
-</Table>
-<br><BR>
-<Table Border Cellspacing="0">
-<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Circle　</Td></Tr>
-<tr><td>　　95℃　</td><td>　2min　</td><Td></Td></tr>
-<Tr><Td>　　95℃　</Td><Td>　15sec　</Td><Td>　25 cycle　</Td></Tr>
-<tr><td>　　59℃　</td><td>　30sec　</td><Td>　25 cycle　</Td></tr><tr>
-<td>　　68℃　</td><td>　1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)　</td><Td>　25 cycle　</Td></tr><tr>
-<td>　　68℃　</td><td>　1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)　</td><Td></Td></tr><tr>
-<td>　　14℃　</td><td>　∞　</td><Td></Td></tr>
-</Table>
-<br><br>
-<img src="https://static.igem.org/mediawiki/2012/6/67/0901bkit.png" width="500" height="300">
 <br><br>
 .	Expected bands for pAct5C DNA and pGaTB DNA were detected on the gel, but band for UAS fragment was not.
@@ Line 222: / Line 196: @@
 <h2>September 4th</h2>
 <br>
-Digestin of GAL4 fragments by XbaⅠ and SpeⅠ
+. Digestion of GAL4 fragments by XbaⅠ and SpeⅠ
 <br>
 <Table Border Cellspacing="0">
-<Tr><Td>　9/3 GAL4　</Td><Td>　40uL　</Td></Tr>
+<Tr><Td>　GAL4 fragments prepared on 9/3　</Td><Td>　40uL　</Td></Tr>
 <tr><td>　M buffer(TOYOBO)　</td><td>　5uL　</td></tr>
 <Tr><Td>　XbaⅠ(TOYOBO)　</Td><Td>　0.5uL　</Td></Tr>
@@ Line 233: / Line 207: @@
 </Table>
 <br><BR>
-Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
+. Digestion of pSB1C3 DNA, LacZ fragments and EGFP fragments with BglⅡ
 <br>
+pSB1C3 DNA prepared on 8/27, LacZ fragment prepared on 9/3 and EGFP fragments were digested with BglⅡ in the following conditions.
+<br>
+<br>
+. Digestion of pSB1C3 DNA with XbaⅠ and SpeⅠ
+<br>
+pSB1C3 DNA (vector) prepared on 8/23 was double digested with XbaⅠ and SpeⅠ.
+<br><br>
 <Table Border Cellspacing="0">
 <Tr><Td>　each DNA　</Td><Td>　40uL　</Td></Tr>
@@ Line 247: / Line 228: @@
 <Table Border Cellspacing="0">
-<Tr><Td>　9/3 GAL4　</Td><Td>　23uL　</Td></Tr>
+<Tr><Td>　pSB1C3 DNA 　</Td><Td>　23uL　</Td></Tr>
 <tr><td>　M buffer(TOYOBO)　</td><td>　10uL　</td></tr>
 <Tr><Td>　XbaⅠ(TOYOBO)　</Td><Td>　1uL　</Td></Tr>
@@ Line 260: / Line 241: @@
 After agarose gel electrophoresis, the digested GAL4 fragments were purified by QIA quick Gel Extraction Kit
 <br><br>
+Photo of agarose gel
+<br>
 <img src="https://static.igem.org/mediawiki/2012/b/b0/0904kit.png" width="500" height="300">
 <br><br>
@@ Line 308: / Line 291: @@
 Ligation was carried out in the following reactions.
-<br><br>
+<br>
 <Table Border Cellspacing="0">
-<Tr><Td></Td><Td>　1uL　</Td></Tr>
+<Tr><Td>　pSB1C3 (XbaⅠand SpeⅠdigested)　</Td><Td>　1uL　</Td></Tr>
-<tr><td></td><td>　1uL　</td></tr>
+<tr><td>　GAL4 (XbaⅠ and SpeⅠ digested)　</td><td>　1uL　</td></tr>
 <Tr><Td>　dH<sub>2</sub>O　</Td><Td>　3uL　</Td></Tr>
 <Tr><Td>　Ligation high　</Td><Td>　2.5uL　</Td></Tr>