Team:TU Delft/20 July 2010 content
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==Salt tolerance== | ==Salt tolerance== | ||
- | Time to get cracking, so bbc1 and B0015 were restricted using the scheme shown below. These were then ligated to one another in the third scheme. | + | Time to get cracking, so bbc1 1T3 and B0015 were restricted using the scheme shown below. These were then ligated to one another in the third scheme. |
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | ||
Line 25: | Line 25: | ||
|1 | |1 | ||
|bbc1 | |bbc1 | ||
- | | | + | |10 |
|- | |- | ||
| | | | ||
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|- | |- | ||
| | | | ||
- | | | + | |Xba1 |
|1 | |1 | ||
|- | |- | ||
Line 41: | Line 41: | ||
| | | | ||
|Water | |Water | ||
- | | | + | |4 |
|- | |- | ||
| | | | ||
Line 49: | Line 49: | ||
|2 | |2 | ||
|B0015 | |B0015 | ||
- | | | + | |10 |
|- | |- | ||
| | | | ||
- | | | + | |Spe1 |
|1 | |1 | ||
|- | |- | ||
| | | | ||
- | | | + | |Pst1 |
|1 | |1 | ||
|- | |- | ||
Line 65: | Line 65: | ||
| | | | ||
|Water | |Water | ||
- | | | + | |4 |
|- | |- | ||
| | | | ||
Line 72: | Line 72: | ||
|- | |- | ||
|3 | |3 | ||
- | | | + | |psb 1T3 |
|1 | |1 | ||
|- | |- | ||
Line 80: | Line 80: | ||
|- | |- | ||
| | | | ||
- | | | + | |Pst1 |
|1 | |1 | ||
|- | |- | ||
| | | | ||
- | |Buffer | + | |Buffer 3 (biolabs) |
|2 | |2 | ||
|- | |- | ||
| | | | ||
|Water | |Water | ||
- | | | + | |6 |
+ | |- | ||
+ | |} | ||
+ | |||
+ | |||
+ | The Ligation (as shown below) was split into two tubes with 25 µl and ligated overnight. | ||
+ | |||
+ | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | ||
+ | |'''#''' | ||
+ | |'''Sample added''' | ||
+ | |'''Volume added (µl)''' | ||
+ | |- | ||
+ | |1 | ||
+ | |psB1T3 | ||
+ | |2 | ||
|- | |- | ||
| | | | ||
- | | | + | |bbc1 |
- | |2 | + | |18 |
+ | |- | ||
+ | | | ||
+ | |B0015 | ||
+ | |18 | ||
+ | |- | ||
+ | | | ||
+ | |Ligase buffer | ||
+ | |5 | ||
+ | |- | ||
+ | | | ||
+ | |T4 DNA Ligase | ||
+ | |2 | ||
+ | |- | ||
+ | | | ||
+ | |Water | ||
+ | |5 | ||
+ | |- | ||
|} | |} | ||
Revision as of 09:40, 10 August 2010
Contents |
Juggling an Diabolo tricks!
From all the pipetting that we do in the lab, our fingers and wrists are a bit tense. After some hard thinking, we found a perfect way to relax them…..by juggling and performing diabolo tricks! So now, in between the incubation times of our experiments the juggle balls are flying around in our iGEM room.
Lab work
Competent cells
Eva made another batch of competent cells
Alkane degradation
BioBrick production continued
A transformation of yesterday's ligation products was done in TOP10 competent cells, and grown on LB-Tetracycline plates overnight. Hopefully we will have some colonies tomorrow.
Salt tolerance
Time to get cracking, so bbc1 1T3 and B0015 were restricted using the scheme shown below. These were then ligated to one another in the third scheme.
# | Sample added | Volume added (µl) |
1 | bbc1 | 10 |
Ecro R1 | 1 | |
Xba1 | 1 | |
Buffer 2 (biolabs) | 2 | |
Water | 4 | |
BSA | 2 | |
2 | B0015 | 10 |
Spe1 | 1 | |
Pst1 | 1 | |
Buffer 2 (biolabs) | 2 | |
Water | 4 | |
BSA | 2 | |
3 | psb 1T3 | 1 |
Ecro R1 | 1 | |
Pst1 | 1 | |
Buffer 3 (biolabs) | 2 | |
Water | 6 |
The Ligation (as shown below) was split into two tubes with 25 µl and ligated overnight.
# | Sample added | Volume added (µl) |
1 | psB1T3 | 2 |
bbc1 | 18 | |
B0015 | 18 | |
Ligase buffer | 5 | |
T4 DNA Ligase | 2 | |
Water | 5 |
Characterization of Anderson RBS sequences
Assembly of reference construct
Single colony PCRs were performed on the transformants obtained yesterday. These were loaded onto 1% agarose gel (see gel below)
Lane description:
# | Description | Expected Length (bp) | Primers | Status | Remarks |
1 | SmartLadder | n/a | n/a | n/a | |
2 | transformant #1 of ligation mix: J23100-B0030-I13401 | G00101 + G00101 | ✗ | ||
3 | transformant #2 of ligation mix: J23100-B0030-I13401 | G00101 + G00101 | ✗ | ||
4 | transformant #3 of ligation mix: J23100-B0030-I13401 | G00101 + G00101 | ✗ | ||
5 | transformant #4 of ligation mix: J23100-B0030-I13401 | G00101 + G00101 | ✗ | ||
6 | transformant #5 of ligation mix: J23100-B0030-I13401 | G00101 + G00101 | ✗ | ||
7 | transformant #1 of ligation mix: I13401 ligation control | G00101 + G00101 | ✗ | ||
8 | transformant #2 of ligation mix: I13401 ligation control | G00101 + G00101 | ✓ | ||
9 | transformant #3 of ligation mix: I13401 ligation control | G00101 + G00101 | ✓ | ||
10 | transformant #1 of ligation mix: I13401 digestion control | G00101 + G00101 | ✓ | ||
11 | transformant #2 of ligation mix: I13401 digestion control | G00101 + G00101 | ✓ | ||
12 | transformant #1 of K081005 | G00101 + G00101 | ✓ | ||
13 | transformant #2 of K081005 | G00101 + G00101 | ✓ | ||
14 | transformant #3 of K081005 | G00101 + G00101 | ✓ | ||
15 | BioRad EZ Load | n/a | n/a | n/a | |
16 | PCR product of E0240 | 1114 | G00101 + G00101 | ✓ | |
17 | PCR product of E0240 | 1114 | G00101 + G00101 | ✓ | |
18 | PCR product of E0240 | 1114 | G00101 + G00101 | ✓ |
Beach
After a hard day working in the lab, we went to the beach in Scheveningen for a nice walk and to watch the sunset.