Team:UNIPV-Pavia/Calendar/July/settimana1

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Revision as of 12:02, 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+ strain	LB, 37°C
BW23474	E. coli pir116 strain	LB, 37°C
BW25141	E. coli pir+ strain	LB, 37°C
BW25142	E. coli pir116 strain	LB, 37°C
BW5328 /pAH123	helper plasmid	LB+Amp LC 30°C,LB+Amp HC 30°C
MC1061	E. coli strain for integration	LB, 37°C
MG165	E. coli wil type strain	LB, 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

All strains received 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.

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 :)

week 1

@@ Line 22: / Line 22: @@
 {| border='1' align='center'
-|| '''name''' || '''description''' || '''growth conditions'''
+|| '''Culture name''' || '''description''' || '''growth conditions'''
 |-
 || BT340 || pCP20 plasmid || LB+Amp LC 30°C,LB+Amp HC 30°C
@@ Line 46: / Line 46: @@
 Cultures were MiniPrepped with the following quantifications (NanoDrop):
 {| border='1' align='center'
-|| '''came''' ||'''quantification'''
+|| '''Culture name''' ||'''quantification'''
 |-
 || I7-1 || 83,3 ng/ul
@@ Line 68: / Line 68: @@
 || PhaP-2 || 13,6 ng/ul
 |}
+All cultures, except PhaP-1 and PhaP-2 were digested. Digestion of:
+{| border="1" align='center'
+|  ''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
+________________________
 [[Image:Unipv_screening_I7_I8_I9_I10.jpg|300px|thumb|center|Screening of I7, I8, I9, I10]]
 All strains received 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.