Team:TU Delft/22 June 2010 content

From 2010.igem.org

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We tried to compensate for yesterdays delays. We transformed 15 Biobricks from the distribution plates!
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=Lab work=
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Some 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.
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==Characterization of Anderson RBS sequences==
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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.  
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The protocol was as follows:
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After analysis on gel we deduced that a temperature of 50 °C was optimal.
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{| style="color:green; background-color:#ffffcc;" cellpadding="10" cellspacing="0" border="1"
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|'''-'''
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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:
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|'''uL'''
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''Sample'''
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|''' Enzyme 1'''
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|'''Enzyme 2'''
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|'''Buffer'''
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|'''BSA'''
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|'''Needed fragment'''
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|-
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|Master Mix
 
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|25
 
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|-
 
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|BB template (10x diluted)
 
|1
|1
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|-
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|1.0 μg pSB1T3
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|Primers G00100 and G00101
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|EcoRI 
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|3
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|PstI
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|-
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|H (Roche)
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|H20
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|✗
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|18
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|‘X–linear pSB1T3–P’
|}
|}
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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.
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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.
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At the end of the day we performed a digestion reaction on the amplified PCR products as well as the previously isolated I13401 and pSB3C5. The general protocol was as follows:
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{| style="color:green; background-color:#ffffcc;" cellpadding="10" cellspacing="0" border="1"
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|Restriction enzyme
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|1 unit per 500 ng DNA
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|-
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|Digestion buffer H
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|2 uL
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|-
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|DNA
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|As deemed necessary
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|-
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|H20
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|to a total volume of 20 uL
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|-
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|}
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Mixtures were incubated at 37 degrees for 2.5 hours and stored at 4 degrees overnight.
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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.

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