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Team:TU Delft/22 June 2010 content
From 2010.igem.org
| Line 4: | Line 4: | ||
The protocol was as follows: | The protocol was as follows: | ||
| - | {| style="color: | + | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" |
| - | | | + | |Component |
| - | | | + | |Sample |
|- | |- | ||
|Master Mix | |Master Mix | ||
| - | |25 | + | |25 μL |
|- | |- | ||
|BB template (10x diluted) | |BB template (10x diluted) | ||
| - | |1 | + | |1 μL |
|- | |- | ||
|Primers G00100 and G00101 | |Primers G00100 and G00101 | ||
| - | |3 | + | |3 μL |
|- | |- | ||
|H20 | |H20 | ||
| - | |18 | + | |18 μL |
|} | |} | ||
| Line 24: | Line 24: | ||
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: | 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: | ||
| - | {| style="color: | + | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" |
|Restriction enzyme | |Restriction enzyme | ||
| - | |1 unit per 1 | + | |1 unit per 1 μg DNA |
|- | |- | ||
|Digestion buffer H | |Digestion buffer H | ||
| - | |2 | + | |2 μL |
|- | |- | ||
|DNA | |DNA | ||
| Line 35: | Line 35: | ||
|- | |- | ||
|H20 | |H20 | ||
| - | |to a total volume of 20 | + | |to a total volume of 20 μL |
|- | |- | ||
|} | |} | ||
Mixtures were incubated at 37 degrees for 2.5 hours and stored at 4 degrees overnight. | Mixtures were incubated at 37 degrees for 2.5 hours and stored at 4 degrees overnight. | ||
Revision as of 11:51, 5 July 2010
We tried to compensate for yesterdays delays. We transformed 15 Biobricks from the distribution plates!
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.
The protocol was as follows:
| Component | Sample |
| Master Mix | 25 μL |
| BB template (10x diluted) | 1 μL |
| Primers G00100 and G00101 | 3 μL |
| H20 | 18 μL |
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.
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:
| Restriction enzyme | 1 unit per 1 μg DNA |
| Digestion buffer H | 2 μL |
| DNA | As deemed necessary |
| H20 | to a total volume of 20 μL |
Mixtures were incubated at 37 degrees for 2.5 hours and stored at 4 degrees overnight.
