Team:Newcastle/Labwork/25 September 2009
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===Introduction=== | ===Introduction=== | ||
+ | * To obtain pspac part from pMutin4, we digested psc fragment with HindIII and BamHI | ||
+ | * We need Mini prep kinA transformation and check the result. | ||
+ | <br> | ||
+ | ===Experiment procedure=== | ||
+ | ==== Digestion ==== | ||
+ | * Digest psc clean-up DNA | ||
- | + | dd H2O 7ul | |
+ | 10X fast digest buffer 7ul | ||
+ | Fast HindIII 3ul | ||
+ | Fast BamHI 3ul | ||
+ | psc clean-up DNA 50ul | ||
+ | ------------------------------ | ||
+ | 70ul | ||
- | === | + | * Incubate 1 hour at 37 degree |
+ | |||
+ | ==== Gel Extraction ==== | ||
+ | * After the digestion of psc fragment we run the digested DNA on big well gel and cut the right band and purified the DNA by gel extraction kit. | ||
+ | |||
+ | ==== Mini prep ==== | ||
- | + | * Followed the Phil's protocal. | |
- | + | * Since we cultured 24 colonies yesterday, we performed Mini Prep for all those 24 strains. | |
- | ==== | + | ==== Test the Mini Prep result ==== |
+ | * Digested the Mini Prep DNA with EcoRI and NheI | ||
+ | dd H2O 7ul | ||
+ | 10X fast digest buffer 2ul | ||
+ | Fast EcoRI 0.5ul | ||
+ | Fast NheI 0.5ul | ||
+ | Mini Prep DNA 10ul | ||
+ | ------------------------------ | ||
+ | 20ul | ||
+ | * 37C incubated for 1 hour | ||
+ | * Add 2ul stop buffer and run the sample on agarose gel. | ||
- | === | + | ==== Ligation ==== |
+ | * Ligate psc fragment with pMutin4 backbone | ||
+ | ddH2O 10ul | ||
+ | ligation buffer 4ul | ||
+ | psc fragments 10ul | ||
+ | pMutin4 backbone (24.9ng/ul) 5ul | ||
+ | T4 ligase 1ul | ||
+ | -------------------------------------- | ||
+ | 30ul | ||
+ | === conclusion === | ||
+ | * After the digestion of kinA transformation Mini Prep result, it seems No.4,5,6,10 could be the right clone. So, we prepare Midi culture for all 4 strains. | ||
<br> | <br> | ||
- | [[Image:|400px|center]] | + | [[Image:Team_newcaslt_2009_hanny_geldoc_250909_1.jpg |400px|center]] |
===Futher plan=== | ===Futher plan=== | ||
+ | * Use HindIII enzyme digest Mini prep result of those 4 strains to double check the result. | ||
{{:Team:Newcastle/Project/Labwork/CalTemplate}} | {{:Team:Newcastle/Project/Labwork/CalTemplate}} |
Latest revision as of 02:53, 21 October 2009
Formal Lab Session - 25th September 2009
Chassis team
Introduction
- To obtain pspac part from pMutin4, we digested psc fragment with HindIII and BamHI
- We need Mini prep kinA transformation and check the result.
Experiment procedure
Digestion
- Digest psc clean-up DNA
dd H2O 7ul 10X fast digest buffer 7ul Fast HindIII 3ul Fast BamHI 3ul psc clean-up DNA 50ul ------------------------------ 70ul
- Incubate 1 hour at 37 degree
Gel Extraction
- After the digestion of psc fragment we run the digested DNA on big well gel and cut the right band and purified the DNA by gel extraction kit.
Mini prep
- Followed the Phil's protocal.
- Since we cultured 24 colonies yesterday, we performed Mini Prep for all those 24 strains.
Test the Mini Prep result
- Digested the Mini Prep DNA with EcoRI and NheI
dd H2O 7ul 10X fast digest buffer 2ul Fast EcoRI 0.5ul Fast NheI 0.5ul Mini Prep DNA 10ul ------------------------------ 20ul
- 37C incubated for 1 hour
- Add 2ul stop buffer and run the sample on agarose gel.
Ligation
- Ligate psc fragment with pMutin4 backbone
ddH2O 10ul ligation buffer 4ul psc fragments 10ul pMutin4 backbone (24.9ng/ul) 5ul T4 ligase 1ul -------------------------------------- 30ul
conclusion
- After the digestion of kinA transformation Mini Prep result, it seems No.4,5,6,10 could be the right clone. So, we prepare Midi culture for all 4 strains.
Futher plan
- Use HindIII enzyme digest Mini prep result of those 4 strains to double check the result.
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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)
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