Team:UNIPV-Pavia/Calendar/July/settimana1

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* '''BW25142''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
* '''BW25142''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
* '''BW25141''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
* '''BW25141''' was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
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METTERE CONDIZIONI DI CRESCITA (temperatura e tempo) e risultati (chi è cresciuto e chi no!)
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==June, 30th==
==June, 30th==

Revision as of 12:12, 5 July 2010

JULY: WEEK 1



June, 28th

Inoculum from glycerol stock for I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 in 5ml LB+Amp. Cultures were gown ON at 37°C 220rpm.

Inoculum of PhaP-1 and PhaP-2 in 5ml LB+Kan and ON growth at 37°C 220rpm.

We received strains and plasmid from ???Yale University???

Culture name description growth conditions
BT340 pCP20 plasmid LB+Amp LC 30°C,LB+Amp HC 30°C
BW23473 E. coli pir+ strainLB, 37°C
BW23474 E. coli pir116 strainLB, 37°C
BW25141 E. coli pir+ strainLB, 37°C
BW25142 E. coli pir116 strainLB, 37°C
BW5328 /pAH123 helper plasmid LB+Amp LC 30°C,LB+Amp HC 30°C
MC1061 E. coli strain for integrationLB, 37°C
MG165 E. coli wil type strainLB, 37°C

June, 29th

All cultures were grown. From I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 5ul were aliquoted and diluted in 500ul LB+Amp for a preliminary TECAN test. Cultures were MiniPrepped with the following quantifications (NanoDrop):

Culture name quantification
I7-1 83,3 ng/ul
I7-2 104 ng/ul
I8-1 123,8 ng/ul
I8-2 103,7 ng/ul
I9-1 117,8 ng/ul
I9-2 110,4 ng/ul
I10-1 98,8 ng/ul
I10-2 93,6 ng/ul
PhaP-1 29,1 ng/ul
PhaP-2 13,6 ng/ul

All cultures, except PhaP-1 and PhaP-2 were digested. Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
I7-1 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I7-2 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I8-1 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I8-2 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I9-1 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I9-2 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I10-1 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5
I10-2 Screening 25 2 19,5 0,5 EcoRI 0,5 PstI 2,5

Digestion was performed at 37°C for 3 hours. Digestions were gel run. For I9 and I10 clones all colonie were correct, while for I7 and I8 extra bands could be observed. Since preliminar results of TECAN test didn't provide encouraging results, we decided to select other 3 colonies both for I7 and I8 and to repeat the screening, while I9 and I10 were correct at this screening, so we choosed I9-1 and I10-1 as definitive I9 and I10 parts. I9 and I10 will be further screened next week.



TECAN TEST



Screening of I7, I8, I9, I10


PhaP-1 and PhaP-2 were prepared or sequencing: 13,8 ul (2x quantity) of PhaP-1 and 14,7ul (1x quantity) oh PhaP-2 were dryed at 65°C and setn to BMR genomics for sequencing service.


All strains received from ???Yale Univesity??? were on blotting paper disks, that were placed on an LB agar plates (with/without antiobiotic, see growth conditions), resuspended wih 80ul LB and then streaked. After plate streaking, disks were trasnferred in falcon containing liquid LB.

  • BT340 was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml).
  • BW5328 was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml).
  • MG1655 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
  • MC1061 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
  • BW25142 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
  • BW25141 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.

METTERE CONDIZIONI DI CRESCITA (temperatura e tempo) e risultati (chi è cresciuto e chi no!)

June, 30th

July, 1st

July, 2nd

Screening of new colonies of I7 and I8. New screening for I9, I10

This time all the clones are OK :)

June

July

week 1

week 2

week 3

week 4