Team:Newcastle/10 August 2010
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==Aims== | ==Aims== | ||
- | We aim to [[Team:Newcastle/Qiagen_Minipreps| extract]] pVeg and lacI from transformed ''E.coli'' DH5alpha (see [[Team:Newcastle/9_August_2010|09.08.2010]])check the concentration of DNA using [[TeamNewcastleNanoDrop_Spectrophotometer| nanodrop]] and perform a [[Team:Newcastle/Restriction_digests|restriction digest]] so we can run the samples on a [[Team:Newcastle/Gel_electrophoresis| gel]] to check the insert sizes. | + | We aim to [[Team:Newcastle/Qiagen_Minipreps| extract]] pVeg and lacI from transformed ''E.coli'' DH5alpha which was cultured in LB broth overnight(see [[Team:Newcastle/9_August_2010|09.08.2010]])check the concentration of DNA using [[TeamNewcastleNanoDrop_Spectrophotometer| nanodrop]] and perform a [[Team:Newcastle/Restriction_digests|restriction digest]] so we can run the samples on a [[Team:Newcastle/Gel_electrophoresis| gel]] to check the insert sizes. |
==Materials and Protocols== | ==Materials and Protocols== | ||
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See protocols for materials | See protocols for materials | ||
+ | |||
+ | ==Results== | ||
+ | |||
+ | Results from Nanodrop: | ||
+ | {|border=1 | ||
+ | |- | ||
+ | ! | ||
+ | !lacI 1 | ||
+ | !lacI 2 | ||
+ | !lacI 3 | ||
+ | !lacI 4 | ||
+ | !pVeg | ||
+ | |- | ||
+ | !Concentration of DNA ng/microlitre | ||
+ | !122.5 | ||
+ | !139.4 | ||
+ | !130.0 | ||
+ | !150.0 | ||
+ | !155.0 | ||
+ | |} | ||
=Subtilin Immunity BioBrick= | =Subtilin Immunity BioBrick= |
Revision as of 13:07, 10 August 2010
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Contents |
Gel extraction of rocF BioBrick components
Gel Electrophoresis for Amplified Pspac_oid promoter, Double terminator and Plasmid Vector pSB1C3
Aim
The aim of the experiment is to perform gel electrophoresis for the amplified fragments of Pspac_oid promoter, double terminator and plasmid vector pSB1C3 and thus perform gel exraction of the required bands.
Materials and Protocol
Please refer to: Gel electrophoresis.
Result
The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution. We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment.
Pspac_oid pormoter | Double Terminator | Plasmid Vector pSB1C3 | |
---|---|---|---|
Size of the Fragment (in bp) | 148 approx. | 116 approx. | 2072 approx. |
Table 1: Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
Discussion
We found two bands in the all lanes out of which one is of approximately of 150 bp is size and the other band is of 80 bp approximately in size.
Conclusion
Gibson assembly of rocF BioBrick
lacI and pVeg
Aims
We aim to extract pVeg and lacI from transformed E.coli DH5alpha which was cultured in LB broth overnight(see 09.08.2010)check the concentration of DNA using nanodrop and perform a restriction digest so we can run the samples on a gel to check the insert sizes.
Materials and Protocols
- Plasmid extraction protocol
- Nanodrop protocol
- Restriction digest protocol
- Gel electrophoresis protocol
- Gel extraction protocol
See protocols for materials
Results
Results from Nanodrop:
lacI 1 | lacI 2 | lacI 3 | lacI 4 | pVeg | |
---|---|---|---|---|---|
Concentration of DNA ng/microlitre | 122.5 | 139.4 | 130.0 | 150.0 | 155.0 |
Subtilin Immunity BioBrick
Aims
Subtilin is required as a cell-signalling molecule in order to trigger calcium carbonate precipitation, as well as bacteria filament formation. We aim to produce 2 subtilin BioBricks; Production and Immunity. Because subtilin is a lantibiotic that is found naturally in Bacillus subtilis ATCC 6633, the introduction of subtilin into our Bacillus 168 cells would actually kill them. Therefore, it is essential to establish immunity for 168 strains.
Materials and Protocol
Please refer to the Phusion PCR protocol.