Team:UNIPV-Pavia/Calendar/July/settimana2

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Revision as of 10:59, 2 August 2010

JULY: WEEK 2

July, 5th

LB agar plates prepared:

LB+Amp 100 (500ml)
LB+Cm 34 (500ml)
LB (250ml)
LB+Cm 12.5 (250ml)

July, 6th

We decided to perform a TECAN test to evaluate the strength of some promoters belonging to the Anderson Promoters Collection. Inoculum from glycerol stpcks (8ul in 5ml LB) for:

<partinfo>BBa_J23101</partinfo>
<partinfo>BBa_J23110</partinfo>
<partinfo>BBa_J23116</partinfo>
<partinfo>BBa_J23118</partinfo>
<partinfo>BBa_J23114</partinfo>
<partinfo>BBa_B0033</partinfo>

TECAN experiment started at 10:20am.

In the morning one single colony was peaked for BW53474 E. coli strain and glycerol stock was prepared after about 7 hours (37°C 220rpm)

Inoculum of:

BW53474 (falcon containing culture for glycerol stock was re-filled with 5ml LB)
MG1655 (from glycerol stock)
BW25141 (from glycerol stock)
BW25142 (from glycerol stock)

July, 7th

Competent cells preparationf for:

MG1655
BW53474
BW25141
BW25142

PCR was performed to amplify Phasins:

File:Phasins PCR.jpg

Phasins amplified by PCR (Marker-Blank-PhaP1-PhaP1-PhaP2-PhaP2)

All parts were correct!! :)

July, 8th

Transformation of 1ul (~4ng) of miniprepped <partinfo>BBa_J23118</partinfo> into 100ul of home-made competent cells

MG1655
BW25141
BW25142
BW53474
DH5-alpha (as control)

to verify the transformation efficiency.

Transformed bacteria were plated on LB+Amp and left overnight in oven, 37°C.

July, 9th

We checked the presence of colonies in plates. All plates showed red colonies!

MG1655 plate (with satellite colonies)	BW25141 plate	BW25142 plate
BW53474 plate	DH5-alpha plate

We calculated (thanks Nicolò) efficiency as #colonies/ug DNA

Strain	#colonies	Efficiency
MG1655	379	~10^5
BW25141	1431	~10^5
BW25142	2374	~10^5
BW53474	9984	~10^6 (very difficult to count correctly)
DH5alpha	7475 (only 100ul of cells plated)	~ 10^6 (very difficult to count correctly, previous tests showed an efficiency of ~10^8)

week 2

@@ Line 78: / Line 78: @@
 |}
-We calculated (thanks Nicolò) efficiency as #colonies/ug DNA plated
+We calculated (thanks Nicolò) efficiency as #colonies/ug DNA
 {|border="1" align="center"
 !Strain || #colonies || Efficiency
@@ Line 84: / Line 84: @@
 | MG1655 || 379 || ~10^5
 |-
-| BW25141 || 1431 || ~10^6
+| BW25141 || 1431 || ~10^5
 |-
-| BW25142 || 2374 || ~10^6
+| BW25142 || 2374 || ~10^5
 |-
-| BW53474 || 9984 || ~10^7<br/>(very difficult to count correctly)
+| BW53474 || 9984 || ~10^6<br/>(very difficult to count correctly)
 |-
-| DH5alpha ||  7475<br/>(only 100ul of cells plated)  || ~ 10^7<br/>(very difficult to count correctly,<br/>previous tests showed an efficiency of ~10^8)
+| DH5alpha ||  7475<br/>(only 100ul of cells plated)  || ~ 10^6<br/>(very difficult to count correctly,<br/>previous tests showed an efficiency of ~10^8)
 |}