Team:TU Delft/22 July 2010 content

From 2010.igem.org

(Difference between revisions)
(Alkane degradation)
(Method 4)
 
(4 intermediate revisions not shown)
Line 117: Line 117:
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.
 +
==Characterisation of Anderson RBS sequences==
-
==Salt tolerance==
+
====Assembly of reference construct====
-
Time to get cracking, so bbc1 and B0015 were restricted using the scheme shown below.
+
=====Method 3=====
 +
 
 +
The Amp plates contained very few transformants. These were taken for [http://2010.igem.org/Team:TU_Delft#page=protocols/colony_PCR single colony PCR] and run on [http://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel 1% agarose gel]:
 +
 
 +
[[Image:TU_Delft_SCpcr_22-07_(pcr_gen).png|450px|thumb|left|1% agarose of colony PCR. Gel run at 100V for 1 hour. Of all samples 5 µL was loaded with 1 µL loadingbuffer. 5 µL was loaded of marker]]
 +
 
 +
Lane description:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|'''Remarks'''
 +
|-
 +
|1
 +
|SmartLadder
 +
|Varies
 +
|n/a
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|2
 +
|Transformant #1 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR)
 +
|1157
 +
|G00100 + G00101
 +
|<font color=red>?</font>
 +
|
 +
|-
 +
|3
 +
|Transformant #2 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR)
 +
|1157
 +
|G00100 + G00101
 +
|<font color=red>?</font>
 +
|
 +
|-
 +
|4
 +
|Transformant #3 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR)
 +
|1157
 +
|G00100 + G00101
 +
|<font color=red>?</font>
 +
|
 +
|-
 +
|5
 +
|Transformant #4 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR)
 +
|1157
 +
|G00100 + G00101
 +
|<font color=red>?</font>
 +
|
 +
|-
 +
|6
 +
|Transformant #5 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR)
 +
|1157
 +
|G00100 + G00101
 +
|<font color=red>?</font>
 +
|
 +
|-
 +
|7
 +
|Transformant #6 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR)
 +
|1157
 +
|G00100 + G00101
 +
|<font color=red>?</font>
 +
|
 +
|-
 +
|8
 +
|PCR product of J23100 in pSB1A2
 +
|1142
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|9
 +
|PCR product of E0240 in pSB1A2
 +
|1114
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|10
 +
|BioRad EZ Load
 +
|Varies
 +
|n/a
 +
|<font color=limegreen>✓</font>
 +
|
 +
|}
 +
 
 +
Due to the very short expected difference in bp between the constructs, the gel could not be conclusive. However, the transformants di not fluoresce when screened with UV excitation.
 +
 
 +
=====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, indicating that the reference construct J23100-E0240 had been created. Three of the colonies were brought over to 5 mL of LB+AMP for over night culturing and characterization.

Latest revision as of 21:56, 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 taken for single colony PCR and run on 1% agarose gel:

1% agarose of colony PCR. Gel run at 100V for 1 hour. Of all samples 5 µL was loaded with 1 µL loadingbuffer. 5 µL was loaded of marker

Lane description:

# Description Expected Length (bp) Primers Status Remarks
1 SmartLadder Varies n/a
2 Transformant #1 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR) 1157 G00100 + G00101 ?
3 Transformant #2 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR) 1157 G00100 + G00101 ?
4 Transformant #3 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR) 1157 G00100 + G00101 ?
5 Transformant #4 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR) 1157 G00100 + G00101 ?
6 Transformant #5 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR) 1157 G00100 + G00101 ?
7 Transformant #6 of ligation mix: S-J23100-pSB1A2-P + X-E0240-P (PCR) 1157 G00100 + G00101 ?
8 PCR product of J23100 in pSB1A2 1142 G00100 + G00101
9 PCR product of E0240 in pSB1A2 1114 G00100 + G00101
10 BioRad EZ Load Varies n/a

Due to the very short expected difference in bp between the constructs, the gel could not be conclusive. However, the transformants di not fluoresce when screened with UV excitation.

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, indicating that the reference construct J23100-E0240 had been created. Three of the colonies were brought over to 5 mL of LB+AMP for over night culturing and characterization.