Team:PKU Beijing/Notebook/AND Gate 1/Core/SupD

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Notebook > AND Gate 1 > Core > Shan Shen's Note

2009.7.23

11:30
Recycle all the inserts of SupD and terminator.

12:20
Digest high-copy bi-stable plasmids.

Total20μL
Plasmids8μL
EcoR11μL
Pst11μL
Buffer2μL
ddH2O8μL

12:30
Start to digest.

12:40
Ligation of SupD+terminator and 1-18A (strongest promoter).

Total10μL
Vectors1μL
Inserts7μL
T4 ligase1μL
Buffer1μL

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

PKU 20090724 Shan Shen 1.JPG

10:30
There are six clones on the plate. Shake those clones in the incubator.
PCR clones to test if they are correct.

Total10μL
TemplateClones
dNTP2μL
Buffer1μL
Taq0.5μL
For-primer0.5μL
Rev-primer0.5μL
ddH2O5.5μL

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 plasmidsConcentration(ng/μL)
Colony 1223
Colony 2355.05
Colony 3148.15
Colony 4124.33
Colony 5113.34
Colony 6146.85

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:

Total20μL
Plasmids2μL
EcoR11μL
Pst11μL
Buffer2μL
ddH2O14μL

Digest into vectors:

Total20μL
Plasmids3μL
EcoR11μL
Pst11μL
Buffer2μL
ddH2O13μL

PCR at the same time:

Total10μL
Template0.5μL
For0.5μL
Rev0.5μL
Mix5μL
ddH2O3.5μL

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:
PKU 20090728 Shan Shen 1.JPG

16:00
Transfrom pKD3, pcp 20 to DH5a.
Digest 1-18C into vectors.

Total20μL
Plasmids3μL
Spe11μL
Pst11μL
Buffer2μL
ddH2O13μL

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.

Total20μL
Plasmids4μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O12μL

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.

Total50μL
Template0.2μL
dNTP4μL
Buffer10μL
Phusion0.5μL
For-primer1.25μL
Rev-primer1.25μL
ddH2O32.8μL

17:30
Digest the high-copy bi-stable PCR products.

Total20μL
Fragments5μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O10μL

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:
PKU 20090729 Shan Shen 1.JPG
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.

Total20μL
Plasmids5μL
EcoR11μL
Kpn11μL
Buffer2μL
ddH2O10μL



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