DATE:8.14
Recorder：Chenyang Li and Weijie Jin
Double Digestion of BD-PYW, AD-PYW-S1, BD-PYW-S1, AD-PYW, AD-PYU and AD-PYU
Procedure:
| number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
| sample |
BD-PYW1001 |
AD-PYW-S11002 |
BD-PYW-S11003 |
AD-PYW1004 |
BD-PYW1005 |
BD-PYW1006 |
AD-PYU1007 |
AD-PYU1008 |
BD-PYW1009 |
| nuclease-free water(μL) |
6.5 |
3.5 |
4 |
2 |
0 |
1.5 |
5 |
1.5 |
0 |
| 10*Green Buffer(μL) |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
| plasmid(μL) |
1.5 |
4.5 |
4.0 |
6 |
8 |
6.5 |
3 |
6.5 |
8 |
| EcoRI(μL) |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
| PstI(μL) |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
Mix gently and incubate at 37 degree Celsius for 15 min.
result:
(from left to right: Trans 2K plus 2(contain Gelred),1001,1,5,6,9,1002,2,1003,3,1004,4,1007,7,1008,8.
Plasmid Extraction for recombinant plasmid pYeT containing sfGFP1-10
Recorder:Yu Xie
Results
All Failed.
Recorder：Chengle Zhang & Yu Xie
Colony picking of recombinant plasmid pYeT containing sfGFP1-10
We did colony picking of combinant plasmid of pYeT and sfGFP1-10.After colony picking,we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.
Date: 8.15
Plasmid Extraction for recombinant plasmid pYeT containing sfGFP1-10
Recorder: Dong Yan & Yu Xie
Sample:bacteria containing recombinant plasmid pYeT containing sfGFP1-10
| Number |
1 |
2 |
3 |
4 |
| A260/A280 |
1.88 |
1.86 |
1.88 |
1.84 |
| concentration(ng/μL) |
741.3 |
639.8 |
655.6 |
602.9 |
Finally succeed, waiting for the PCR results.
Recorder: Yu Xie
PCR of supernatant which have recombinant plasmid pYeT containing sfGFP1-10
Procedure:
1.Prepare 4 PCR tubes and sequentially add：
| sample |
1,2,3,4 |
| Sterilized ddH2O |
9 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template(supernatant) |
1.5 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
1 min |
| Anneal |
58 |
1 min |
| Extend |
72 |
1 min 10s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Agarose gel electrophoresis Results:
Lane 14,15,16,17
Failed
Recorder: Yu Xie
PCR of bacteria which have recombinant plasmid pYeT containing sfGFP1-10
Procedure:
1.Prepare 4 PCR tubes and sequentially add：
| sample |
1,2,3,4 |
| Sterilized ddH2O |
9 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template(bacteria) |
1.5 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
1 min |
| Anneal |
58 |
1 min |
| Extend |
72 |
1 min 10s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Agarose gel electrophoresis Results:
Lane 10,11,12,13
Finally succeed!!!
Recorder: Yu Xie
PCR of recombinant plasmid pYeT containing sfGFP1-10
Procedure:
1.Prepare 4 PCR tubes and sequentially add：
| sample |
1,2,3,4 |
| Sterilized ddH2O |
9.5 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template(plasmid) |
1 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
1 min |
| Anneal |
58 |
1 min |
| Extend |
72 |
1 min 10s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Agarose gel electrophoresis Results:
Lane 6,7,8,9
Finally succeed!!!
Recorder:Chengle Zhang
Plasmid Extraction for YeUGAP(0222)
| Number |
1 |
2 |
3 |
4 |
5 |
| A260/A280 |
1.89 |
1.88 |
1.87 |
1.88 |
1.87 |
| concentration(ng/μL) |
649.4 |
718.8 |
698.2 |
148.0 |
452.7 |
Plasmid Extraction for YeUGAP
Recorder: Yinchenguang Lyu
| Number |
1 |
2 |
3 |
4 |
5 |
| A260/A280 |
1.86 |
1.86 |
1.87 |
1.87 |
1.81 |
| A260/A230 |
2.33 |
2.16 |
2.32 |
2.28 |
1.73 |
| concentration(ng/μL) |
716.2 |
480.6 |
485.1 |
381.5 |
599.9 |
Recorder: Chenyang Li
PCR of bacteria which have recombinant plasmid containing sup35-sfGFP11
Procedure:
- Prepare 4 PCR tubes and sequentially add：
| sample |
08151001 |
08151002 |
08151003 |
08151004 |
| Sterilized ddH2O |
8.5μL |
8μL |
8.5μL |
8μL |
| 2×HiFi-PCR Master |
10μL |
10μL |
10μL |
10μL |
| Template(bacteria) |
0.5 μL |
1μL |
0.5 μL |
0.5 μL |
| Primer 1 |
0.5 μL |
0.5 μL |
0.5 μL |
0.5 μL |
| Primer 2 |
0.5 μL |
0.5 μL |
0.5 μL |
0.5 μL |
| total |
20 μL |
- PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
50 s |
| Anneal |
55 |
1 min |
| Extend |
72 |
1 min 20 s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Recorder: Chenyang Li
PCR of bacteria which have recombinant plasmid PSB1A3 containing promoter+RBS+sfGFP1-10
Procedure:
- Prepare 4 PCR tubes and sequentially add：
| sample |
08151005 |
08151006 |
08151007 |
08151008 |
| Sterilized ddH2O |
8.8μL |
8.8μL |
8.8μL |
8.8μL |
| 2×HiFi-PCR Master |
10μL |
10μL |
10μL |
10μL |
| Template(bacteria) |
0.2μL |
|
0.2μL |
|
0.2μL |
|
0.2μL |
| Primer 1 |
0.5 μL |
0.5 μL |
0.5 μL |
0.5 μL |
| Primer 2 |
0.5 μL |
0.5 μL |
0.5 μL |
0.5 μL |
| total |
20 μL |
- PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
50 s |
| Anneal |
55 |
1 min |
| Extend |
72 |
1 min 20 s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Recorder：Chenyang Li
Double Digestion of YeUGAP
Procedure:
| number |
08151009 |
08151010 |
08151011 |
08151012 |
08151013 |
| nuclease-free water(μL) |
7.2 |
7.2 |
7.2 |
6.7 |
7.2 |
| 10*Green Buffer(μL) |
1 |
1 |
1 |
1 |
1 |
| plasmid(μL) |
1 |
1 |
1 |
1.5 |
1 |
| EcoRI(μL) |
0.6 |
0.6 |
0.6 |
0.6 |
0.6 |
| PstI(μL) |
0.2 |
0.2 |
0.2 |
0.2 |
0.2 |
Mix gently and incubate at 37 degree Celsius for 15 min.
result:
(from left to right: Trans 2K plus 2(contain Gelred), contral 0801,08151009,08151010,08151011,08151012,08151013,contral 0805)
Recorder: Yonghao Liang
Transformation of plasmid YeWGAP
PS:After we cultivate the bacteria for 10 hours, we take 800 μL of bacteria out from one tube into 400 μL glycerinum. And put all the tubes into fridge.
Recorder:Chengle Zhang
Plasmid Extraction for ADU
| Number |
ADU3-1 |
ADU3-2 |
ADU3-3 |
ADU4-1 |
ADU4-2 |
ADU4-3 |
| A260/A280 |
1.89 |
1.91 |
1.90 |
1.94 |
1.87 |
1.92 |
| concentration(ng/μL) |
261.8 |
204.8 |
208.6 |
98.5 |
121.4 |
120.9 |
DATE:8.16
Recorder:Chengle Zhang and Yinchenguang Lyu
PCR of AD and BD
We did 4 tubes of AD, 4 of BD.
|
|
| 22μL |
Sterilized ddH2O |
| 25μL |
2×raq PCR Master Mix |
| 1μL |
template |
| 2μL |
primer 1 |
| 2μL |
primer 2 |
| 50μL |
total |
time set
| stage |
temperature |
time |
| Pre-Duration |
94 |
4 min |
| Duration |
94 |
30 s |
| Anneal |
60 |
30 s |
| Extend |
72 |
45 |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
50 s |
| Anneal |
55 |
1 min |
| Extend |
72 |
1 min 20 s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Recorder: Yinchenguang Lyu
purification of the product of PCR
results
| Sample |
AD1 |
AD2 |
BD1 |
BD2 |
| A260/A280 |
1.86 |
1.86 |
1.85 |
1.87 |
| A260/A230 |
2.25 |
2.18 |
2.04 |
2.36 |
| concentration(ng/μL) |
338.1 |
257.0 |
336.0 |
301.4 |
Recorder: Chengle Zhang
Purification of AD and BD from PCR
Results:
| Sample |
AD1 |
AD2 |
BD1 |
BD2 |
| A260/A280 |
1.88 |
1.87 |
1.87 |
1.87 |
| concentration(ng/μL) |
197.8 |
184.6 |
273.6 |
262.8 |
Double digestion of AD,BD and PYU
Recorder:Chengle Zhang
| Sample |
AD1(0228) |
AD2 |
BD1(0229) |
BD2 |
PYU1(0230) |
PYU2 |
| A260/A280 |
1.39 |
- |
1.83 |
1.99 |
1.90 |
1.86 |
| concentration(ng/μL) |
189.6 |
- |
44.3 |
26.1 |
80.8 |
121.5 |
Recorder：Ya Jiang
Ligation of sup35 and pUC57-sfGFP11
Reaction system:
3 μL pUC57-sfGFP11,
2 μL 10× T4 DNA ligase buffer,
0.2 μL T4 DNA ligase,
15 μL sup35
Mix gently and incubate at 16 degree Celsius overnight.
Recorder：Ya Jiang
Ligation of AD and PYU
Reaction system:
3 μL PYU,
2 μL 10× T4 DNA ligase buffer,
0.2 μL T4 DNA ligase,
15 μL AD
Mix gently and incubate at 22 degree Celsius for an hour.
*Recorder:Chenyang Li
**Plasmid Extraction for YeWGAP, ADU and BDW **
| Number |
YeWGAP5 |
YeWGAP6 |
ADU1 |
ADU2 |
BDW3 |
BDW4 |
| A260/A280 |
1.81 |
1.70 |
1.89 |
1.65 |
1.82 |
1.87 |
| A260/A230 |
0.63 |
0.75 |
2.21 |
0.79 |
1.45 |
2.10 |
| concentration(ng/μL) |
41.3 |
14.4 |
44.8 |
124.5 |
39.8 |
46.7 |
Recorder: Chenyang Li and Weijie Jin
Transformation of plasmid YeWGAP
Designed by 2016 iGEM Team:USTC
Under CC License
Based on Semantic-UI
