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Team:Groningen/Labwork/15 July 2013
From 2013.igem.org
(Created page with ""Sander" <br/>made MC 10x according to protocol B.Subtilis transformation except instead of adding H2O till it 10ml, 10ml total was added.") |
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<br/>made MC 10x according to protocol B.Subtilis transformation except instead of adding H2O till it 10ml, 10ml total was added. | <br/>made MC 10x according to protocol B.Subtilis transformation except instead of adding H2O till it 10ml, 10ml total was added. | ||
| + | |||
| + | '''Mirjam''' | ||
| + | <br/> Made PCR reactions to obtain a higher silk concentration, but analysis revealed that the expected bands are not present. An attempt to concentrate the friday samples failed for 3 of the 4 tubes. | ||
| + | |||
| + | Because the transformation failed again last week. This time a new restriction and ligation is made for the promoter and the backbone. A new protocol for transformation is used. | ||
| + | <br/>- tubes are prechilled on ice | ||
| + | <br/>- competent ''E.coli'' cells are defrosted on ice | ||
| + | <br/>- 10 ul ligation reaction is added to the competent cells, or 2 ul backbone for the positive control | ||
| + | <br/>- incubation on ice for 30 min | ||
| + | <br/>- heat shock on 42 degrees Celsius for 1 min | ||
| + | <br/>- incubation on ice for 5 min | ||
| + | <br/>- add 1 ml of LB medium | ||
| + | <br/>- incubation at 37 degrees Celsius for 1 hour | ||
| + | <br/>- centrifugation and resuspend the pellet in 200 ul. | ||
| + | <br/>- plate on Lb agar + ampicillin | ||
| + | <br/>- incubate O/N at 37 degrees Celsius | ||
Revision as of 12:06, 15 July 2013
Sander
made MC 10x according to protocol B.Subtilis transformation except instead of adding H2O till it 10ml, 10ml total was added.
Mirjam
Made PCR reactions to obtain a higher silk concentration, but analysis revealed that the expected bands are not present. An attempt to concentrate the friday samples failed for 3 of the 4 tubes.
Because the transformation failed again last week. This time a new restriction and ligation is made for the promoter and the backbone. A new protocol for transformation is used.
- tubes are prechilled on ice
- competent E.coli cells are defrosted on ice
- 10 ul ligation reaction is added to the competent cells, or 2 ul backbone for the positive control
- incubation on ice for 30 min
- heat shock on 42 degrees Celsius for 1 min
- incubation on ice for 5 min
- add 1 ml of LB medium
- incubation at 37 degrees Celsius for 1 hour
- centrifugation and resuspend the pellet in 200 ul.
- plate on Lb agar + ampicillin
- incubate O/N at 37 degrees Celsius
