Team:KIT-Kyoto/Notebook-week3p

From 2012.igem.org

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<h2>September 6th</h2>
 
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Transformation performed on September 5th was not successful, since we had no colony on the plate.
 
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<br><br>
 
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1. PCR amplification of UAS , UAS-TNFAIP3, pSB1C3DNA
 
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<br>
 
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<Table Border Cellspacing="0">
 
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<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr>
 
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<tr><td> 10× KOD plus buffer </td><td> 5uL </td></tr>
 
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<Tr><Td> 2mM dNTPs </Td><Td> 5uL </Td></Tr>
 
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<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 1.6uL </Td></Tr><tr>
 
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<td> 10P 5’ primer </td><td> 1.5uL </td></tr>
 
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<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr>
 
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<Tr><td> KOD plus </td><td> 1uL </td></tr>
 
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<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr>
 
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<Tr><td> Total </td><td> 50uL </td></tr>
 
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</Table>
 
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Reaction conditions
 
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<Table Border Cellspacing="0">
 
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<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr>
 
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<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
 
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<Tr><Td>  95℃ </Td><Td> 15sec</Td><Td> 30 cycle </Td></Tr>
 
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<tr><td>  58℃ </td><td> 30sec</td><Td> 30 cycle </Td></tr><tr>
 
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<td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr>
 
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<tr><td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr>
 
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<tr><td>  14℃ </td><td> ∞ </td><Td></Td></tr>
 
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</Table>
 
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2. PCR products were applied to the agarose gel electrophoresis
 
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<br>
 
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From left to right: UAS, UAS-TNFAIP3, pSB1C3
 
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<br><br>
 
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3. Purifucation of pSB1C3 PCR products
 
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<br>
 
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pSB1C3 PCR products were purified from the gel by QIA quick Gel Extraction Kit.
 
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<br><br>
 
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<img src="http://2012.igem.org/wiki/images/5/53/0906kit.png" width="500" height="300">
 
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<br><br>
 
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4. Ligation of pSB1C3 DNA and GAL4 fragment
 
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<br>
 
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<Table Border Cellspacing="0">
 
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<Tr><Td> pSB1C3(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr>
 
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<Tr><Td> GAL4(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr>
 
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<Tr><td> dH<sub>2</sub>O </td><td> 3uL </td></tr>
 
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<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr>
 
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<Tr><td> Total </td><td> 7.5uL </td></tr>
 
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</Table>
 
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<br><BR>
 
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5. The ligation products were transformed into E. coli XL-1and spread on the LB Chloramphenicol(+) plate
 
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<br><br>
 
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<h2>September 7th</h2>
<h2>September 7th</h2>
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<br>

Revision as of 02:55, 26 September 2012






September 7th


1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA
 DNA template  0.25uL 
 10× KOD plus buffer  5uL 
 2mM dNTPs  5uL 
 25mM MgSO4  1.6uL 
 10P 5’ primer  1.5uL 
 10P 3’ primer  1.5uL 
 KOD plus  1uL 
 dH2O  34.15uL 
 Total  50uL 


Reaction conditions
 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  58℃  2min30sec  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS)  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS) 
  14℃  ∞ 




2. PCR products were applied to the agarose gel electrophoresis
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below
Results: no clearly amplified bands were detected.

3. Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6

4. SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL


From these results, concentration of the pSB1C3 DNA was estimated to be 10ng/uL.

GAL4
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker 2uL, DNA 0.1uL, 1kbmarker 1uL



However no GAL DNA was detected. We lost the sample somewhere by some mistakes.

September 8th


1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA
 DNA template  1uL 
 10× KOD plus buffer  5uL 
 2mM dNTPs  5uL 
 25mM MgSO4  1.6uL 
 10P 5’ primer  1.5uL 
 10P 3’ primer  1.5uL 
 KOD plus  1uL 
 dH2O  33.4uL 
 Total  50uL 


Reaction conditions
 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  58℃  2min30sec  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS)  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS) 
  14℃  ∞ 


5. PCRproducts applied to the agarose gel electrophoresis
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each


Only the PCR products for pSB1C3 was detectable.
We repeated PCR for UAS and GAL4DNA by changing the annealing temperature
(-1 indicates 55℃、-2 indicates 58℃)

 DNA template  1uL 
 10× KOD plus buffer  5uL 
 2mM dNTPs  5uL 
 25mM MgSO4  1.6uL 
 10P 5’ primer  1.5uL 
 10P 3’ primer  1.5uL 
 KOD plus  1uL 
 dH2O  33.4uL 
 Total  50uL 


Reaction conditions
 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  55℃(-1) or 58℃(-2)  2min30sec  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS)  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS) 
  14℃  ∞ 


September 9th


1. PCRproducts were electrophoreses.
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each


We finally succeeded the amplification of the UAS fragments.

2. Purification of pSB1C3 and UAS-1-PCR products
PCR products were purified by High Pure PCR Product Kit

3. The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction

 UAS  40uL 
 4 buffer(NEB)  5uL 
 EcoRⅠ-HF(NEB)  0.5uL 
 SpeⅠ(NEB  0.5uL 
 100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 


4. The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit



5. PCR amplification of GAL4
 DNA template  1uL 
 10× KOD plus buffer  5uL 
 2mM dNTPs  5uL 
 25mM MgSO4  1.6uL 
 10P 5’ primer  1.5uL 
 10P 3’ primer  1.5uL 
 KOD plus  1uL 
 dH2O  33.4uL 
 Total  50uL 


Reaction conditions
 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  58℃  2min30sec  30 cycle 
  68℃  2min10sec  30 cycle 
  68℃  2min10sec 
  14℃  ∞ 


6. PCR products were applied to the agarose gel electrophoresis as shown below.


7. pSB1C3 and UAS fragments were ligated in the following reactions.
 pSB1C3 (EcoRⅠ and SpeⅠ digested)  1uL 
 UAS (EcoRⅠ and SpeⅠ digested)  1uL 
 dH2O  3uL 
 Ligation high  2.5uL 
 Total  7.5uL 


The following reaction were carried out as a negative control.
 pSB1C3 (EcoRⅠ and SpeⅠ digested) 1uL 
 dH2O  4uL 
 Ligation high  2.5uL 
 Total  7.5uL 


8. 7 ligation products were transformed into E. coli XL-1 Blue and spread on the LB Chloramphenicol(+) plate and incubated at 37℃ for 16 hours.

September 10th


At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.

1. Single colony isolation of ligationproducts
Ligation product prepared on 9/9 (UAS and pSB1C3) gave us some colonies and negative control sample gave us no colony. We therefore picked up four independent colonies and cultured them in 2.5mL LB Chloramphenicol (+) medium at 37℃ for 16 hours.

2. Digestion of HS promoter fragment and Act5C promoter with XbaⅠ and BamHⅠ
DNA fragments carrying HS promoter (prepared on 9/5) and those carrying Act5C promoter (prepared on 9/3) were digested with XbaⅠ and BamHⅠ.

 HS or Act5c  40uL 
 4 buffer(NEB)  5uL 
 XbaⅠ-HF(NEB)  0.5uL 
 BamHⅠ(NEB ) 0.5uL 
 100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 


Digested fragments were purified from the gel by QIA quick Gel Extraction Kit.

Photo of agarose gel


3. PCR amplification of GAL4 fragments
We tried PCR under four different conditions described below.

 G-1 and G-2  G-3 and G-4 
 DNA template  1uL  4uL 
 10× KOD plus buffer  5uL  5uL 
 2mM dNTPs  5uL  5uL 
 25mM MgSO4  1.6uL  1.6uL 
 10P 5’ primer  1uL  1uL 
 10P 3’ primer  1uL  1uL 
 KOD plus  1uL  1uL 
 dH2O  33.4uL  31.4uL 
 Total  50uL  50uL 


Reaction conditions
 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  57℃(G-1 and G-3) or 59℃(G-2 and G-4)  2min30sec  30 cycle 
  68℃  2min10sec  30 cycle 
  68℃  2min10sec 
  14℃  ∞ 


4. PCR products were applied to agarose gel electrophoresis
From left to right: 10uL each G-1, G-2, G-3, G-4

Photo of agarose gel


We found that GAL4 sequence was properly amplified under G-1 and G-2conditions. 5. PCR products were purified by High Pure PCR Product Kit.
6. Digestion of pSB1C3 DNA with EcoRⅠ and SpeⅠ
PCR-amplified pSB1C3 DNA was digested with EcoRⅠ and SpeⅠ for 2 hours at 37℃.

 pSB1C3(PCR)  40uL 
 4 buffer(NEB)  5uL 
 EcoRⅠ-HF(NEB)  0.5uL 
 SpeⅠ(NEB)  0.5uL 
 2100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 


7. Digestion of EcoRI-digested UAS fragment by BglⅡ
BglⅡ-digestion was carried out for 2 hours at 37℃ under conditions described below.

 UAS(EcoRⅠ-digested)  13uL 
 3 buffer(NEB)  2uL 
 BglⅡ(NEB)  0.5uL 
 dH2O  2.5uL 
 Total  20uL 


8. Purification of restriction enzyme-digested pSB1C3 DNA and UAS fragment
restriction enzyme-digested pSB1C3 DNA and UAS fragment (prepared on 9/10) were purified by QIA quick Gel Extraction Kit.

Photo of agarose gel


9. PCR-amplified GAL4 fragments were digested with XbaⅠ and SpeⅠ under conditions described below.

 GAL4  20uL 
 4 buffer(NEB)  4uL 
 XbaⅠ(NEB)  0.7uL 
 SpeⅠ(NEB)  0.7uL 
 100×BSA  0.4uL 
 dH2O  14.6uL 
 Total  40uL 


The digested DNA was applied to agarose gel electrophoresis as shown below.

Photo of agarose gel


Results: XbaⅠ-digestion of GAL4 fragment gave two DNA fragments, indicating that GAL4 sequence contains XbaⅠ site.

10. Digestion of GAL4 sequence with BglⅡ and SpeⅠ
Digestion was carried out st 37℃ for 1hour under the conditions described below.
 GAL4  40uL 
 3 buffer(NEB)  5uL 
 BglⅡ(NEB)  0.5uL 
 dH2O  4.5uL 
 Total  50uL 


The digested DNA was purified by High Pure PCR Product Kit was further digested with Spe I at 37℃ for 1hour.

 GAL4(Bgl Ⅱ cut)  40uL 
 4 buffer(NEB)  5uL 
 SpeⅠ  0.5uL 
 100×BSA  0.5uL 
 dH2O  4uL 
 Total  50uL 


The digested fragments were purified by QIA quick Gel Extraction Kit.

Photo of agarose gel


11. Insertion of GAL4 fragments and HS promoter or Act5C promoter-enhancer and UASfragment and EGFP sequence or LacZ sequence into the pSB1C3DNA

Ligation reactions were carried out at 16℃ for 1hour under conditions described below.

 pSB1C3 (PCRproducts)(double digested with EcoRⅠ and SpeⅠ)  1uL 
 UAS (double digested with EcoRⅠ and BglⅡ)  2uL 
 EGFP or LacZ  2uL 
 Ligation high  5uL 
 Total  10uL 


 pSB1C3 (vector DNA) (double digested with XbaⅠ and SpeⅠ)  1uL 
 GAL4 (double digested with BglⅡ and SpeⅠ)  2uL 
HS or Act5c (double digested with XbaⅠ and BamHⅠ) 2uL 
 Ligation high  5uL 
 Total  10uL 


12. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.

September 11th


1. Single colony isolation
Single colonies were isolated from the plate prepared on 9/11 and cultured in 2.5mL LB Chloramphenicol(+) medium at 37℃ for 16 hours.

2. Isolation of the candidate pSB1C3-UAS DNA
The candidate pSB1C3-UAS DNA was purified by QIA prep Spin Miniprep Kit.

3. The purified candudate pSB1C3-UAS was digested with BglII for 2 hours.

 pSB1C3-UAS-1, -2, -3, -4  5uL 
 3buffer(NEB)  2uL 
 BglⅡ  0.5uL 
 dH2O  12.5uL 
 Total  20uL 


The digested samples were applied to agarose gel electrophoresis as shown below. Left to right: pSB1C3-1 uncut, pSB1C3 cut, -2 uncut, -2 cut, -3 uncut, -3 cut, -4 uncut, -4 cut

Photo of agarose gel


Results: All DNAs examined had a single BglII site, indicating that the pSB1C3-UAS DNA was successfully constructed.

The BglII-digested pSB1C3-UAS DNA was purified from the gel by QIA quick Gel Extraction Kit.

4. Digestion of pSB1C3 (PCR) with XbaⅠ and SpeⅠ
PCR-amplified pSB1C3 DNA was double digested with XbaⅠ and SpeⅠ at 37℃ for 2 hours.

 GAL4  40uL 
 4 buffer(NEB)  5uL 
 XbaⅠ(NEB)  0.5uL 
 SpeⅠ(NEB)  0.5uL 
 CIP  0.5uL 
 100×BSA  0.5uL 
 dH2O  3uL 
 Total  40uL 


The digested samples were purified by QIA quick Gel Extraction Kit

Photo of agarose gel


5. Insertion of GAL4, HS promoter or Act5C promoter-enhancer into the pSB1C3DNA

Ligation reactions were carried out under conditions described below at 16℃ for 1hour .
 pSB1C3 (vector) (digested with XbaⅠ and SpeⅠ)  1uL 
 GAL4 (digestion with BglⅡ and SpeⅠ)  2.5uL 
 HS promoter or Act5C promoter-enhancer (digested with XbaⅠ and BamHⅠ)  2.5uL 
 Ligation high  6uL 
 Total  12uL 


 pSB1C3 (PCR products) (digested with XbaⅠ and SpeⅠ)  2uL 
 GAL4 (digested with BglⅡ and SpeⅠ)  2.5uL 
 HS promoter or Act5C promoter-enhancer (XbaⅠ and BamHⅠ)  1.5uL 
 Ligation high  6uL 
 Total  12uL 


13. Ligation products were transformed into E. coli Xl-1 blue, spread on the LB Chloramphenicol(+) plate and cultured at 37℃for 16 hours.

September 12th


1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs
We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11).

1-1-47 Digestion of pSB1C3-UAS DNA by Spe1
We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.

Composition
 pSB1C3-UAS-1, -2  40uL 
 3buffer(NEB)  5uL 
 SpeⅠ  1uL 
 100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 


The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.

Photo of Agarose gel.


1-1-48 and 2-2-6 Ligation
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.

Ligation reactions are listed below.
 pSB1C3-UAS (double digested with BglII and SpeⅠ)  1uL 
 EGFP fragments or LacZ fragments  2uL 
 EGFP or LacZ  2uL 
 Ligation high  2.5uL 
 dH2O  2uL 
 Total  7.5uL 


We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.

Composition
 pSB1C3 (PCR) (double digested with XbaⅠand SpeI)  2uL 
 GAL4(double digested with BglⅡ and SpeⅠ)  2uL 
 HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ)  2uL 
 Ligation high  6uL 
 Total  12uL 


1-1-49 and 2-2-7 Transformation of E. coli by Ligation products
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.