Team:TU Delft/22 July 2010 content

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A number of colonies look promising! To check if they really are the BioBricks we want, tomorrow we will do a plasmid isolation with the cultures of lane 2, 4, 6, 7, 9 and 14. We will cut the isolated plasmids with various restriction enzymes and analyze the digestion products on gel.
A number of colonies look promising! To check if they really are the BioBricks we want, tomorrow we will do a plasmid isolation with the cultures of lane 2, 4, 6, 7, 9 and 14. We will cut the isolated plasmids with various restriction enzymes and analyze the digestion products on gel.
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==Characterisation of Anderson RBS sequences==
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====Assembly of reference construct====
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=====Method 3=====
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The Amp plates contained very few transformants. These were [http://2010.igem.org/Team:TU_Delft#page=protocols/colony_PCR single colony PCR'd] and run on [http://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel 1% agarose gel]:
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=====Method 4=====
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After having stored yesterday's Amp plates in the fridge it was decided to screen once more for GFP fluorescence. This time multiple fluorescing colonies were found under UV excitation. Three of the colonies were brought over to 5 mL of LB+AMP for over night culturing and characterization.

Revision as of 21:22, 16 August 2010

Contents

Lab work

Alkane degradation

There were some colonies on Tuesday's plates! We had left the plates @ 37°C yesterday after having seen that there were no colonies. When checking this morning on all plates (except the negative control) there were a few colonies! (2-50 colonies). Chances are it's not what we're looking for, but maybe they are good transformants... to check we will do a colony PCR.

1% Agarose gel of colony PCR. Gel runned at 100V for 1 hour. Of all samples 5 μL + 1 μL loadingbuffer was loaded. 5 μL was loaded of marker.
# Description Expected length (bp) Primers Status
1 SmartLadder n/a n/a n/a
2 Transformant #1 of ligation mix 007T 1616 G00100 + G00101
3 Transformant #2 of ligation mix 007T 1616 G00100 + G00101
4 Transformant #3 of ligation mix 007T 1616 G00100 + G00101
5 Transformant #1 of ligation mix 008T 551 G00100 + G00101
6 Transformant #2 of ligation mix 008T 551 G00100 + G00101
7 Transformant #1 of ligation mix 009T 551 G00100 + G00101
8 Transformant #1 of ligation mix 009T 560 G00100 + G00101
9 Transformant #1 of ligation mix 009T 560 G00100 + G00101
10 Transformant #1 of ligation mix 010T 1657 G00100 + G00101
11 Transformant #1 of ligation mix 010T 1657 G00100 + G00101
12 Transformant #1 of ligation mix 010T 1657 G00100 + G00101
13 Transformant #1 of ligation mix 017T 1130 G00100 + G00101
14 Transformant #1 of ligation mix 017T 1130 G00100 + G00101
15 Transformant #1 of ligation mix 017T 1130 G00100 + G00101
16 Transformant #1 of ligation mix 018T 1874 G00100 + G00101
17 Transformant #1 of Red colony 1360 G00100 + G00101

A number of colonies look promising! To check if they really are the BioBricks we want, tomorrow we will do a plasmid isolation with the cultures of lane 2, 4, 6, 7, 9 and 14. We will cut the isolated plasmids with various restriction enzymes and analyze the digestion products on gel.

Characterisation of Anderson RBS sequences

Assembly of reference construct

Method 3

The Amp plates contained very few transformants. These were single colony PCR'd and run on 1% agarose gel:


Method 4

After having stored yesterday's Amp plates in the fridge it was decided to screen once more for GFP fluorescence. This time multiple fluorescing colonies were found under UV excitation. Three of the colonies were brought over to 5 mL of LB+AMP for over night culturing and characterization.