Team:KIT-Kyoto/Notebook-week3p

From 2012.igem.org

(Difference between revisions)
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<h2>September 7th</h2>
<h2>September 7th</h2>
<br>
<br>
-
1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA
+
<strong>1-1-33 and 2-1-23 PCR amplification of UAS, UAS-TNFAIP3, pSB1C3, GAL4 DNA</strong>
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
-
2. PCR products were applied to the agarose gel electrophoresis
+
<strong>1-1-34 and 2-1-24 PCR products were applied to the agarose gel electrophoresis</strong>
<br>
<br>
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 as shown below
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<br><br>
<br><br>
Results: no clearly amplified bands were detected.
Results: no clearly amplified bands were detected.
 +
<br>
 +
<br>Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6
<br><br>
<br><br>
-
3. Re-estimation of the concentration of pSB1C3 and GAL4 DNA used for ligation on 9/5, 6
+
<strong>2-1-25 SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis</strong>
-
<br><br>
+
-
4. SB1C3 and GAL4 DNA were applied to the agarose gel electrophoresis
+
<br>
<br>
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL
From left to right: 1kbmarker 5uL, DNA 1uL, 1kb marker 3uL, DNA 0.2uL, 1kbmarker - 2uL, DNA 0.1uL, 1kbmarker 1uL
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<h2>September 8th</h2>
<h2>September 8th</h2>
<br>
<br>
-
1. PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA
+
<strong>1-1-35 and 2-1-26</strong>
 +
<br>PCR amplification of UAS, UAS-TNFAIP3, pSB1C3 and GAL4 DNA
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
-
2. PCRproducts applied to the agarose gel electrophoresis
+
<strong>1-1-36 and 2-1-27 PCRproducts applied to the agarose gel electrophoresis</strong>
<br>
<br>
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each
From left to right: UAS, UAS-TNFAIP3, pSB1C3, GAL4 10 uL each
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<br>
<br>
<br>
<br>
-
3. We repeated PCR for UAS and GAL4DNA by changing the annealing temperature
+
<strong>1-1-37 and 2-1-28</strong>
 +
<br>
 +
We repeated PCR for UAS and GAL4DNA by changing the annealing temperature
<br>
<br>
（-1 indicates 55℃、-2 indicates 58℃）
（-1 indicates 55℃、-2 indicates 58℃）
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<h2>September 9th</h2>
<h2>September 9th</h2>
<BR>
<BR>
-
1. PCRproducts were electrophoreses.
+
<strong>1-1-38 and 2-1-29</strong>
 +
<br>
 +
PCRproducts were electrophoreses.
<br>
<br>
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each
Left to right: UAS-1, UAS-2, GAL4-1, GAL4-2 10 uL each
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We finally succeeded the amplification of the UAS fragments.
We finally succeeded the amplification of the UAS fragments.
<br><br>
<br><br>
-
2. Purification of pSB1C3 and UAS-1-PCR products
+
<strong>1-1-39 Purification of pSB1C3 and UAS-1-PCR products</strong>
<br>
<br>
PCR products were purified by High Pure PCR Product Kit
PCR products were purified by High Pure PCR Product Kit
<br><br>
<br><br>
-
3. The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction
+
<strong>1-1-40 </strong>
 +
<br>
 +
The purified UAS fragments were digested with EcoRⅠ and SpeⅠ in the following reaction
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
-
4. The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit
+
<strong>1-1-41</strong>
 +
<br>
 +
The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit
<br><br>
<br><br>
<img src="https://static.igem.org/mediawiki/2012/9/91/0909bkit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/9/91/0909bkit.png" width="500" height="300">
<br><br>
<br><br>
-
5. PCR amplification of GAL4
+
<strong>2-1-30 PCR amplification of GAL4</strong>
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
-
6. PCR products were applied to the agarose gel electrophoresis as shown below.
+
<strong>2-1-31</strong>
 +
<br>
 +
PCR products were applied to the agarose gel electrophoresis as shown below.
<br>
<br>
<img src="https://static.igem.org/mediawiki/2012/b/bb/0909ckit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/b/bb/0909ckit.png" width="500" height="300">
<br><br>
<br><br>
-
7. pSB1C3 and UAS fragments were ligated in the following reactions.
+
<strong>1-1-42</strong>
 +
<br>
 +
pSB1C3 and UAS fragments were ligated in the following reactions.
<br>
<br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
-
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.
+
<strong>1-1-43</strong>
 +
<br>
 +
Ligation products(1-1-42) were transformed into E. coli XL-1 Blue  and spread on the LB Chloramphenicol(＋) plate and incubated at 37℃ for 16 hours.
<br><br>
<br><br>

Revision as of 06:40, 26 September 2012

September 7th


1-1-33 and 2-1-23 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℃　　∞　


1-1-34 and 2-1-24 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.

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

2-1-25 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


1-1-35 and 2-1-26
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℃　　∞　


1-1-36 and 2-1-27 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.

1-1-37 and 2-1-28
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


1-1-38 and 2-1-29
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.

1-1-39 Purification of pSB1C3 and UAS-1-PCR products
PCR products were purified by High Pure PCR Product Kit

1-1-40
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　


1-1-41
The digested UAS DNA was applied to the agarose gel electrophoresis and purified from the gel by QIA quick Gel Extraction Kit



2-1-30 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℃　　∞　


2-1-31
PCR products were applied to the agarose gel electrophoresis as shown below.


1-1-42
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　


1-1-43
Ligation products(1-1-42) 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℃.

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