"
Team:Newcastle/Labwork/25 September 2009
From 2009.igem.org
(Difference between revisions)
(→conclusion) |
|||
| (5 intermediate revisions not shown) | |||
| Line 5: | Line 5: | ||
[[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]] | [[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]] | ||
=Formal Lab Session - 25th September 2009= | =Formal Lab Session - 25th September 2009= | ||
| + | ==<u> Chassis team </u>== | ||
| + | |||
| + | ===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> | ||
| + | [[Image:Team_newcaslt_2009_hanny_geldoc_250909_1.jpg |400px|center]] | ||
| + | |||
| + | ===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/Footer}} | {{:Team:Newcastle/Footer}} | ||
{{:Team:Newcastle/Right}} | {{:Team:Newcastle/Right}} | ||
Latest revision as of 02:53, 21 October 2009
Project
Sub-projects
Wet Lab
Meetings
Planning
Links
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.
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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]
