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Team:Warsaw/Calendar-Stage1/11 July 2010
From 2010.igem.org
Monday, 12 June 2010;
2. Digest fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins:
- GFP E240
- CFP E0420
- YFP E0430
- mOrange E2050*
- mCherry J67102*
- mCherry NLS I712028
* can have an internal RBS. Check by cloning behind J23100.
3. Gel electrophoresis:
Samples in following order: E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
4. Gel extraction and ligation of fluorescent proteins with expression vector.
Not all samples at expected size, some vectors at wrong size.
Looks like order of some samples is mixed. We will check it **after** transformation, because those are fluorescent proteins therefe it is easy to find the correct samples. Vector with J23100 of expexted size.
______________________________________________
Digest conditions, used for all reactions:
8 μl DNA
2 μl BamHI buffer
10 μl H2O
0,3 μl enzymów
20 μl final volume
digest for 3h
______________________________________________
Ligation conditions, used for all reactions:
25ul insert DNA
2ul vector DNA
3ul ligase buffer
30 ul final volume
overnight ligation
Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100
1. Digest part part 3.1 – Anderson RBS from distribution (expression vector = promoter J23100+RBS) with SpeI and PstI
2. Digest fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins:
- GFP E240
- CFP E0420
- YFP E0430
- mOrange E2050*
- mCherry J67102*
- mCherry NLS I712028
* can have an internal RBS. Check by cloning behind J23100.
3. Gel electrophoresis:
Samples in following order: E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
4. Gel extraction and ligation of fluorescent proteins with expression vector.
GEL
Samples: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
Not all samples at expected size, some vectors at wrong size.
Looks like order of some samples is mixed. We will check it **after** transformation, because those are fluorescent proteins therefe it is easy to find the correct samples. Vector with J23100 of expexted size.
______________________________________________
Digest conditions, used for all reactions:
8 μl DNA
2 μl BamHI buffer
10 μl H2O
0,3 μl enzymów
20 μl final volume
digest for 3h
______________________________________________
Ligation conditions, used for all reactions:
25ul insert DNA
2ul vector DNA
3ul ligase buffer
30 ul final volume
overnight ligation
