Team:TU Delft/22 June 2010 content

From 2010.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 1: Line 1:
-
===Lab Work===
+
=Lab work=
-
We tried to compensate for yesterdays delays. We transformed 15 Biobricks from the distribution plates!
+
==Characterization of Anderson RBS sequences==
 +
We tried to compensate for yesterdays delays. Thias performed a [[Team:TU_Delft/protocols/colony_PCR|PCR reaction]] on short BioBrick inserts of J61100, J61101, J61107, J61117, J61127 and B0034. We wanted to investigate if this would save time in the long-run compared to transforming and plasmid isolation. The PCR reaction was run at varying annealing temperatures to figure out which temperature gave the best result.
-
Some [[Team:TU_Delft/protocols/PCR|PCR]] reactions were performed on short BioBrick inserts which would eventually save time in the long-run (compared to transforming and plasmid isolation). The BioBricks involved were J61100, J61101, J61107, J61117, J61127 and B0034.
+
After analysis on gel we deduced that a temperature of 50 °C was optimal.
-
BB template (10x diluted)
+
At the end of the day we performed a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|Restriction digestion reaction]] on the amplified PCR products as well as the previously isolated I13401 and pSB3C5:
-
The PCR reaction was run at varying annealing temperatures to figure out which temperature gave the best result. After analysis on gel we deduced that a temperature of 50 degrees Celsius was optimal.
+
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Sample'''
 +
|''' Enzyme 1'''
 +
|'''Enzyme 2'''
 +
|'''Buffer'''
 +
|'''BSA'''
 +
|'''Needed fragment'''
 +
|-
 +
|1
 +
|1.0 μg pSB1T3
 +
|EcoRI 
 +
|PstI
 +
|H (Roche)
 +
|✗
 +
|‘X–linear pSB1T3–P’
 +
|}
-
At the end of the day we performed a [[Team:TU_Delft/protocols/restriction_enzyme_digestion|Restriction digestion reaction]] on the amplified PCR products as well as the previously isolated I13401 and pSB3C5.  
+
In stead of 10 U/μL restriction enzyme we used 1 U/μL. Mixtures were incubated at 37 °C for 2.5 hours and stored at 4 °C overnight.
-
 
+
-
Mixtures were incubated at 37 degrees for 2.5 hours and stored at 4 degrees overnight.
+

Latest revision as of 19:50, 5 August 2010

Lab work

Characterization of Anderson RBS sequences

We tried to compensate for yesterdays delays. Thias performed a PCR reaction on short BioBrick inserts of J61100, J61101, J61107, J61117, J61127 and B0034. We wanted to investigate if this would save time in the long-run compared to transforming and plasmid isolation. The PCR reaction was run at varying annealing temperatures to figure out which temperature gave the best result.

After analysis on gel we deduced that a temperature of 50 °C was optimal.

At the end of the day we performed a Restriction digestion reaction on the amplified PCR products as well as the previously isolated I13401 and pSB3C5:

# Sample Enzyme 1 Enzyme 2 Buffer BSA Needed fragment
1 1.0 μg pSB1T3 EcoRI PstI H (Roche) ✗ ‘X–linear pSB1T3–P’

In stead of 10 U/μL restriction enzyme we used 1 U/μL. Mixtures were incubated at 37 °C for 2.5 hours and stored at 4 °C overnight.

Retrieved from "http://2010.igem.org/Team:TU_Delft/22_June_2010_content"