"
Team:Newcastle/Labwork/23 September 2009
From 2009.igem.org
(Difference between revisions)
(→Stochastic switch team) |
(→Stochastic Switch Team) |
||
| (13 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 - 23rd September 2009= | =Formal Lab Session - 23rd September 2009= | ||
| - | ==<u>Stochastic | + | [[Image:Team Newcastle 2009 iGEM 23-09-09 IMG 1358.JPG|350px|center]] |
| - | + | <br> | |
| + | =<font color="Orange"><u>Overview</u></font>= | ||
| + | <font color="Orange"> | ||
| + | *[[#Stochastic Switch Team|Stochastic Switch Team]] '''- ''' | ||
| + | <br> | ||
| + | *[[#Sporulation Tuning/Chassis Team|Sporulation Tuning/Chassis Team]] '''- ''' | ||
| + | <br> | ||
| + | </font> | ||
| + | <br> | ||
| + | ==<u>Stochastic Switch Team</u>== | ||
| + | [[Image:Team Newcastle 2009 iGEM 23-09-09 IMG 1346.JPG|250px|center]] | ||
Today we discovered that the transfrormations seemed sucessful, and the control plates had no growth at all which is what was hoped for. Hopefuly this means that we have a transformed ara-sspb-pSB1AT3 ligation product. | Today we discovered that the transfrormations seemed sucessful, and the control plates had no growth at all which is what was hoped for. Hopefuly this means that we have a transformed ara-sspb-pSB1AT3 ligation product. | ||
* We set up miniprep cultures for 12 colonies using LB + amp + tet | * We set up miniprep cultures for 12 colonies using LB + amp + tet | ||
| - | * We also set up 12 more miniprep cultures for the Sac transformed cells in hope that the precious minipreps had been carried out incorrectly. | + | * We also set up 12 more miniprep cultures for the Sac transformed cells in hope that the precious minipreps had been carried out incorrectly. |
| + | <br> | ||
| + | |||
| + | <br> | ||
| + | |||
| + | ==<u>Sporulation Tuning/Chassis Team</u>== | ||
| + | [[Image:Team Newcastle 2009 iGEM 23-09-09 IMG 1340.JPG|200px|right]] | ||
| + | ===Introduction=== | ||
| + | * Today's main work is to ligate kinA with pGFP-rrnB vector and transform ligated vector into E.coli. | ||
| + | |||
| + | <br> | ||
| + | |||
| + | ===Experiment procedure=== | ||
| + | |||
| + | ==== ligate kinA and pGFP-rrnB ==== | ||
| + | * ligate reaction | ||
| + | dd H2O 14.5ul | ||
| + | Fast ligation buffer 4ul | ||
| + | pGFP-rrnB backbone(180ng/ul) 0.5ul | ||
| + | kinA fragment(6.1ng/ul) 15ul | ||
| + | T4 ligase 1ul | ||
| + | --------------------------------------------- | ||
| + | 35ul | ||
| + | |||
| + | * Votex for few seconds. | ||
| + | * 22C 10min and 1 hour in RT. | ||
| + | |||
| + | ==== Digest pMK-RQ:kinA and pGFP-rrnB ==== | ||
| + | * pMK-RQ digest reaction. | ||
| + | dd H2O 7ul | ||
| + | 10X fast digest buffer 7ul | ||
| + | Fast EcoRI 3ul | ||
| + | Fast NheI 3ul | ||
| + | pMK-RQ vector 50ul | ||
| + | ------------------------------ | ||
| + | 70ul | ||
| + | * pGFP-rrnB digest reaction. | ||
| + | dd H2O 7ul | ||
| + | 10X fast digest buffer 7ul | ||
| + | Fast EcoRI 3ul | ||
| + | Fast NheI 3ul | ||
| + | pGFP-rrnB vector 50ul | ||
| + | ------------------------------ | ||
| + | 70ul | ||
| + | |||
| + | * Incubate 1 hour at 37 degree. | ||
| + | |||
| + | ==== Purify the digest fragment and backbone ==== | ||
| + | [[Image:Team Newcastle 2009 iGEM 23-09-09 IMG 1368.JPG|200px|right]] | ||
| + | * Run the digested DNA on big well agarose gel. | ||
| + | * cut the right bands. | ||
| + | * Use gel extraction kit to purify the DNA fragments. | ||
| + | |||
| + | ==== Transformation ==== | ||
| + | * Followed the Phil's transformation protocal. | ||
| + | |||
| + | ==== Make LB+Spectinomycin plates==== | ||
| + | * The spectinomycin solution in frige is 20mg/ml | ||
| + | * The concentration needed in plate is 50ug/ml | ||
| + | * Add 2.5ml spectinomycin solution into 1 liter LB agar medium and pour the plate | ||
| + | |||
| + | <br> | ||
| + | [[Image:|400px|center]] | ||
| + | |||
| + | ===Further plan=== | ||
| + | * If th transformation was success, we need to prepare the Mini and Midi culture. | ||
| + | * Collect the plasmid with kinA and do transformation for B.subtilis. | ||
| + | |||
| + | {{:Team:Newcastle/Project/Labwork/CalTemplate}} | ||
{{:Team:Newcastle/Footer}} | {{:Team:Newcastle/Footer}} | ||
{{:Team:Newcastle/Right}} | {{:Team:Newcastle/Right}} | ||
Latest revision as of 15:41, 21 October 2009
Project
Sub-projects
Wet Lab
Meetings
Planning
Links
Formal Lab Session - 23rd September 2009
Overview
Stochastic Switch Team
Today we discovered that the transfrormations seemed sucessful, and the control plates had no growth at all which is what was hoped for. Hopefuly this means that we have a transformed ara-sspb-pSB1AT3 ligation product.
- We set up miniprep cultures for 12 colonies using LB + amp + tet
- We also set up 12 more miniprep cultures for the Sac transformed cells in hope that the precious minipreps had been carried out incorrectly.
Sporulation Tuning/Chassis Team
Introduction
- Today's main work is to ligate kinA with pGFP-rrnB vector and transform ligated vector into E.coli.
Experiment procedure
ligate kinA and pGFP-rrnB
- ligate reaction
dd H2O 14.5ul
Fast ligation buffer 4ul
pGFP-rrnB backbone(180ng/ul) 0.5ul
kinA fragment(6.1ng/ul) 15ul
T4 ligase 1ul
---------------------------------------------
35ul
- Votex for few seconds.
- 22C 10min and 1 hour in RT.
Digest pMK-RQ:kinA and pGFP-rrnB
- pMK-RQ digest reaction.
dd H2O 7ul
10X fast digest buffer 7ul
Fast EcoRI 3ul
Fast NheI 3ul
pMK-RQ vector 50ul
------------------------------
70ul
- pGFP-rrnB digest reaction.
dd H2O 7ul
10X fast digest buffer 7ul
Fast EcoRI 3ul
Fast NheI 3ul
pGFP-rrnB vector 50ul
------------------------------
70ul
- Incubate 1 hour at 37 degree.
Purify the digest fragment and backbone
- Run the digested DNA on big well agarose gel.
- cut the right bands.
- Use gel extraction kit to purify the DNA fragments.
Transformation
- Followed the Phil's transformation protocal.
Make LB+Spectinomycin plates
- The spectinomycin solution in frige is 20mg/ml
- The concentration needed in plate is 50ug/ml
- Add 2.5ml spectinomycin solution into 1 liter LB agar medium and pour the plate
[[Image:|400px|center]]
Further plan
- If th transformation was success, we need to prepare the Mini and Midi culture.
- Collect the plasmid with kinA and do transformation for B.subtilis.
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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]
