"
Team:Newcastle/Labwork/8 October 2009
From 2009.igem.org
(Difference between revisions)
(→Formal Lab Session - 8th October 2009) |
|||
| (6 intermediate revisions not shown) | |||
| Line 2: | Line 2: | ||
{{:Team:Newcastle/Header}} | {{:Team:Newcastle/Header}} | ||
{{:Team:Newcastle/Left}} | {{:Team:Newcastle/Left}} | ||
| - | + | __NOTOC__ | |
[[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]] | [[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]] | ||
=Formal Lab Session - 8th October 2009= | =Formal Lab Session - 8th October 2009= | ||
| - | Today we did restriction | + | == Introduction == |
| - | been contaminated. We ordered normal new enzymes and used them instead. | + | *Today we did restriction digests again with ''pMUTIN4'' with the new enzymes. We concluded that the enzymes we used may have been contaminated. We ordered normal new enzymes and used them instead. |
| + | *We also did PCR fragment digests (from the cleaned PCR product) with BamHI and HindIII | ||
| + | * We run the digestions on the gel and cut PCR fragment and pmutin4 from the gel to clean up. | ||
| + | * Our B.subtilis transformation worked well yesterday, so we can perform new transformation using our pGFP-rrnB:kinA vector. | ||
| + | * Also we set up mini and midi culture yesterday, we need to do mini prep and midi prep today. | ||
| - | pmutin4 digest with HindIII and BamHI | + | == Experiment procedure == |
| + | ===pmutin4 digest === | ||
| + | 1.pmutin4 digest with HindIII and BamHI | ||
H2O 26ul | H2O 26ul | ||
10X Buffer E 5ul | 10X Buffer E 5ul | ||
| Line 17: | Line 23: | ||
BamHI 1ul | BamHI 1ul | ||
| - | pmutin4 control digest with HindIII | + | 2.pmutin4 control digest with HindIII |
H2O 13.5 ul | H2O 13.5 ul | ||
10X Buffer E 1ul | 10X Buffer E 1ul | ||
| Line 24: | Line 30: | ||
HindIII 0.5ul | HindIII 0.5ul | ||
| - | pmutin4 control digest with BamHI | + | 3.pmutin4 control digest with BamHI |
H2O 13.5 ul | H2O 13.5 ul | ||
10X Buffer E 1ul | 10X Buffer E 1ul | ||
| Line 31: | Line 37: | ||
BamHI 0.5ul | BamHI 0.5ul | ||
| - | pSB1AT3 control digest with HindIII and BamHI | + | 4.pSB1AT3 control digest with HindIII and BamHI |
H2O 13 ul | H2O 13 ul | ||
10X Buffer E 1ul | 10X Buffer E 1ul | ||
| Line 39: | Line 45: | ||
BamHI 0.5ul | BamHI 0.5ul | ||
| - | + | === B.subtilis transformation === | |
| + | |||
| + | * Follow the protocal from our lab protocol of B.subtilis transformation. | ||
| + | * 5ul kinA DNA was used for transformation. | ||
| + | |||
| + | === Midi prep cotC part === | ||
| + | * After the standared Midi procedure, we performed DNA concentration process and suspended midi DNA in 250ul PCR water. | ||
| - | + | === Mini prep cotC and kinA culture === | |
| + | * Followed Phil's Mini prep protocal. | ||
{{:Team:Newcastle/Project/Labwork/CalTemplate}} | {{:Team:Newcastle/Project/Labwork/CalTemplate}} | ||
{{:Team:Newcastle/Footer}} | {{:Team:Newcastle/Footer}} | ||
{{:Team:Newcastle/Right}} | {{:Team:Newcastle/Right}} | ||
Latest revision as of 17:37, 21 October 2009
Project
Sub-projects
Wet Lab
Meetings
Planning
Links
Formal Lab Session - 8th October 2009
Introduction
- Today we did restriction digests again with pMUTIN4 with the new enzymes. We concluded that the enzymes we used may have been contaminated. We ordered normal new enzymes and used them instead.
- We also did PCR fragment digests (from the cleaned PCR product) with BamHI and HindIII
- We run the digestions on the gel and cut PCR fragment and pmutin4 from the gel to clean up.
- Our B.subtilis transformation worked well yesterday, so we can perform new transformation using our pGFP-rrnB:kinA vector.
- Also we set up mini and midi culture yesterday, we need to do mini prep and midi prep today.
Experiment procedure
pmutin4 digest
1.pmutin4 digest with HindIII and BamHI
H2O 26ul 10X Buffer E 5ul BSA 0.5 ul (diluted) DNA 15ul HindIII 1ul BamHI 1ul
2.pmutin4 control digest with HindIII
H2O 13.5 ul 10X Buffer E 1ul BSA 0.5 ul (diluted) DNA 3ul HindIII 0.5ul
3.pmutin4 control digest with BamHI
H2O 13.5 ul 10X Buffer E 1ul BSA 0.5 ul (diluted) DNA 3ul BamHI 0.5ul
4.pSB1AT3 control digest with HindIII and BamHI
H2O 13 ul 10X Buffer E 1ul BSA 0.5 ul (diluted) DNA 3ul HindIII 0.5ul BamHI 0.5ul
B.subtilis transformation
- Follow the protocal from our lab protocol of B.subtilis transformation.
- 5ul kinA DNA was used for transformation.
Midi prep cotC part
- After the standared Midi procedure, we performed DNA concentration process and suspended midi DNA in 250ul PCR water.
Mini prep cotC and kinA culture
- Followed Phil's Mini prep protocal.
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
News
Events
- 20 – 21 June 2009 - Europe workshop (London)
- 23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
- 23 October Practice Presentation (Newcastle)
- 23 October T-shirts are ready
- 27 October Practice Presentation (Sunderland)
- 27 October Poster is ready
- 30 October – 2 November 2009 - Jamboree (Boston)
Social Net
- Newcastle iGEM Twitter
- [http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
- [http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]
