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Team:Groningen/Notebook/Wetwork 23July2012
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expected size: 3300bp for the pSB1C3 (2079bp)+ alsT (~500bp)+ GFP (800bp) | expected size: 3300bp for the pSB1C3 (2079bp)+ alsT (~500bp)+ GFP (800bp) | ||
| - | From the gel picture, only plasmid from colony C2 (promoter alsT300) showed the expected size. The other plasmids seem to be empty, probably due to self ligation | + | From the gel picture, only plasmid from colony C2 (promoter alsT300) showed the expected size. The other plasmids seem to be empty, probably due to self ligation. |
Solution: Check plasmid from other white colony | Solution: Check plasmid from other white colony | ||
| - | + | re do transformation. Increase the ratio of plasmid backbone: insert gene | |
| + | |||
| + | |||
| + | Building backbone biobrick for B.subtilis | ||
| + | |||
| + | plasmid pSac-Cm: integration plasmid, double cross over in SacA region | ||
| + | |||
| + | add double terminator, BBa_B0015, and prefix-suffix: XbaI, SpeI, PstI | ||
| + | |||
| + | |||
| + | How to? | ||
| + | |||
| + | Cut backbone with HindIII and EcoRI | ||
| + | |||
| + | Cut BBa_B0015 with HindIII and EcoRI | ||
| + | |||
| + | Now we have separate parts ready to use for ligation into our biobrick device | ||
Revision as of 13:47, 25 July 2012
Tom and Arjan started with gas chromatography mass spectrometry. Test sample: 5gr. fresh meat incubated @ 37C for 3 houres. For both column (HP5 and CP wax) three runs on each column with different incubation and syringe temperature (1: 80C, 100C 2: 100C, 120C 3: 120C, 140C)
Nisa
Plasmid isolation pSB1C3-alsT promoter + GFP
NanoDrop result (ng/ul):
A1= 102.9
A2= 96,6
B1= 118,1
B2= 113,7
C1= 122,8
C2= 88 Comment: gel picture OK!
D1= 124,9
D2= 93,3
Result:
expected size: 3300bp for the pSB1C3 (2079bp)+ alsT (~500bp)+ GFP (800bp)
From the gel picture, only plasmid from colony C2 (promoter alsT300) showed the expected size. The other plasmids seem to be empty, probably due to self ligation.
Solution: Check plasmid from other white colony
re do transformation. Increase the ratio of plasmid backbone: insert gene
Building backbone biobrick for B.subtilis
plasmid pSac-Cm: integration plasmid, double cross over in SacA region
add double terminator, BBa_B0015, and prefix-suffix: XbaI, SpeI, PstI
How to?
Cut backbone with HindIII and EcoRI
Cut BBa_B0015 with HindIII and EcoRI
Now we have separate parts ready to use for ligation into our biobrick device
