(Difference between revisions)
(August 2)
Line 1: Line 1:
==August 2==
==August 2==
Line 139: Line 140:
=Golden Gate Assembly=
==August 10==
==August 10==
Golden Gate Assembly method
Golden Gate Assembly method

Revision as of 06:01, 28 August 2012




August 2

Mutation of FT by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.

Inverse PCR
10xBufer2mM dNTPsprimer fwdprimer revtemplatepolymeraseMilliQTotal

94°C 2min, (98°C 10sec, 68°C 4min)x4cycles, 4°C Hold

Dpn1 Digestion
PCR productDpn1
37°C,1h incubate
productMilliQLigaseT4 KinaseTotal

16°C, 1h incubate

competent cellDNA

Cells were stored on ice for 30min.
After 42°C 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37°C for 1hr, plated to Kanamycin plate.

August 13

Liquid culture of FT 37°C, overnight

August 14

Miniprep of FT : by Sato, Takeuchi
The concentrarion was 81.5ng/uL
Restriction digestion and Electrophoresis : by Sato
To check wheter mutation was succeed, we did restriction enzyme digestion.


37°C 2h incubate
We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.
However, we couldn't get any bands (data not shown.)

Liquid culture of FT (4mL)

August 15

Miniprep of FT : by Sato, Takeuchi, Hyungcheol
We couldn't get enough concentration of plasmids.
Electrophoresis : by Sato
We retried electrophoresis of three samples same as yesterday.
However, we couldn't get any bands as well.

August 16

Transformation : by Takeuchi, Ota

NameWellSampleCompetent CellsTotalPlateColony
FT-1 µL1011LB (Kan+)×
pSB1C31-3-A11011LB (CP+)
I7190051-15-N11011LB (Amp+)

August 17

Transformation : by Takeuchi

NameWellSampleCompetent CellsMilliQTotalPlateColony
FT-221822LB (Kan+)×

We found that mutation of FT was not successful.

August 20

We decided to do PCR using FT specific primers before mutation.
PCR of FT  : by Sato

10xBuferdNTPsMgSO4primer fwdprimer revtemplatepolymeraseMilliQTotal
553111(130ng/µL)1(KOD plus neo)3350

94°C 2min, (98°C 10sec, 68°C 15sec)x30cycles, 4°C Hold
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 203ng/µL.

11kb ladder-

August 21

Restriction digestion  : by Sato


37°C, 5h incubate
After the ethanol precipitation, we diluted in 30µL of MilliQ.
The concentration was 54,9ng/µL.

Ligation  : by Sato

VectorInsertLigation High Ver.2

Liquid culture
T7 promoter, pSB1C3 (4mL)

August 22

Miniprep : by Sato

T7 promoterpSB1C3

Golden Gate Assembly

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Safety Attributions