2009.7.23
11:30
Recycle all the inserts of SupD and terminator.
12:20
Digest high-copy bi-stable plasmids.
| Total | 20μL
|
| Plasmids | 8μL
|
| EcoR1 | 1μL
|
| Pst1 | 1μL
|
| Buffer | 2μL
|
| ddH2O | 8μL
|
12:30
Start to digest.
12:40
Ligation of SupD+terminator and 1-18A (strongest promoter).
| Total | 10μL
|
| Vectors | 1μL
|
| Inserts | 7μL
|
| T4 ligase | 1μL
|
| Buffer | 1μL
|
16:50
Electrophoresis to recycle the high-copy bi-stable digestion products.
The order and the amount of the samples: control plasmids 2μL, digestion products, 20μL marker 5μL.
2009.7.24
10:30
There are six clones on the plate. Shake those clones in the incubator.
PCR clones to test if they are correct.
| Total | 10μL
|
| Template | Clones
|
| dNTP | 2μL
|
| Buffer | 1μL
|
| Taq | 0.5μL
|
| For-primer | 0.5μL
|
| Rev-primer | 0.5μL
|
| ddH2O | 5.5μL
|
The results are not very good.
14:00
Transform the plasmids PKD46 from France in two tubes.
22:40
Miniprep 1-18A + SupD + terminator.
| Number of the plasmids | Concentration(ng/μL)
|
| Colony 1 | 223
|
| Colony 2 | 355.05
|
| Colony 3 | 148.15
|
| Colony 4 | 124.33
|
| Colony 5 | 113.34
|
| Colony 6 | 146.85
|
2009.7.28
00:20
Digest 1-18A + SupD +terminator plasmids to test if they are correct. Digest them into vectors as well.
Digest into inserts:
| Total | 20μL
|
| Plasmids | 2μL
|
| EcoR1 | 1μL
|
| Pst1 | 1μL
|
| Buffer | 2μL
|
| ddH2O | 14μL
|
Digest into vectors:
| Total | 20μL
|
| Plasmids | 3μL
|
| EcoR1 | 1μL
|
| Pst1 | 1μL
|
| Buffer | 2μL
|
| ddH2O | 13μL
|
PCR at the same time:
| Total | 10μL
|
| Template | 0.5μL
|
| For | 0.5μL
|
| Rev | 0.5μL
|
| Mix | 5μL
|
| ddH2O | 3.5μL
|
1:00
Start to digest.
1:30
Start to PCR.
12:00
Electrophoresis to test if the PCR products and digestion products are correct.
The order and the of the samples: marker, PCR products 1, PCR 2, PCR 3, PCR4, PCR5, PCR6, digestion control plasmids, digestion1, digestion2, digestion3, digestion4, digestion5, digestion 6.
Results:
16:00
Transfrom pKD3, pcp 20 to DH5a.
Digest 1-18C into vectors.
| Total | 20μL
|
| Plasmids | 3μL
|
| Spe1 | 1μL
|
| Pst1 | 1μL
|
| Buffer | 2μL
|
| ddH2O | 13μL
|
20:00
Electrophoresis the digestion products:
The order and the amount of the samples: marker 10μL, plasmids control 2μL, digestion 1 15μL, digestion2 15μL.
Results:
The digestion products are correct.
2009.7.29
10:00
Recycle the digestion products.
Miniprep the plasmids of 1-18C and PYFP.
13:30
Digest plasmids PYFP from France.
| Total | 20μL
|
| Plasmids | 4μL
|
| EcoR1 | 1μL
|
| Kpn1 | 1μL
|
| Buffer | 2μL
|
| ddH2O | 12μL
|
13:48
Start to digest.
15:00
Transform psp20 into DH5a.
Ligation of 1-18C backbone and SupD+ terminator inserts.
15:30
PCR to standardize the PYFP plasmids.
Take a gradient from 54 centigrade to 58 centigrade.
| Total | 50μL
|
| Template | 0.2μL
|
| dNTP | 4μL
|
| Buffer | 10μL
|
| Phusion | 0.5μL
|
| For-primer | 1.25μL
|
| Rev-primer | 1.25μL
|
| ddH2O | 32.8μL
|
17:30
Digest the high-copy bi-stable PCR products.
| Total | 20μL
|
| Fragments | 5μL
|
| EcoR1 | 1μL
|
| Kpn1 | 1μL
|
| Buffer | 2μL
|
| ddH2O | 10μL
|
19:30
Electrophoresis the digestion products and PCR products.
The order of the samples: marker, plasmids control, digestion products, PCR products under 54 centigrade, PCR products under 56 centigrade, PCR products under 58 centigrade.
Results:
Digestion products are weird, while the PCR products under 58 centigrade are specialized. As a result, recycle the PCR products under 58 centigrade.
21:55
Digest PYFP plasmids.
| Total | 20μL
|
| Plasmids | 5μL
|
| EcoR1 | 1μL
|
| Kpn1 | 1μL
|
| Buffer | 2μL
|
| ddH2O | 10μL
|
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