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Team:Newcastle/Labwork/10 September 2009
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__NOTOC__ | __NOTOC__ | ||
| - | =Lab | + | [[Image:Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG|50px|right]] |
| - | ==Metal | + | =Formal Lab Session - 10th September 2009= |
| + | [[Image:Team Newcastle 2009 iGEM 10-09-09 IMG 1053.JPG|400px|center]] | ||
| + | <br> | ||
| + | =<font color="Orange"><u>Overview</u></font>= | ||
| + | <font color="Orange"> | ||
| + | *[[#Metal Sensor Team|Metal Sensor Team]] '''- ''' | ||
| + | <br> | ||
| + | *[[#Stochastic Switch Team|Stochastic Switch Team]] '''- ''' | ||
| + | <br> | ||
| + | *[[#Sporulation Tuning/Chassis Team|Sporulation Tuning/Chassis Team]] '''- ''' | ||
| + | <br> | ||
| + | </font> | ||
| + | <br> | ||
| + | ==<u>Metal Sensor Team</u>== | ||
===Introduction=== | ===Introduction=== | ||
| + | Yesterday saw the Metal Sensing team attempt to transform ''DH5-alpha E.coli'' bacterial cells with two BioBricks (the ''cotC-GFP-smtA'' BioBrick and the ''kinA'' BioBrick) and plate them out on LB + kanamycin plates. The ''cotC-GFP-smtA'' BioBrick is part of the Metal Sensing team's sub-project and the ''kinA'' BioBrick will be used by the Stochastic Switch team's sub-project. | ||
| + | |||
| + | '''Today's exercise should see the Metal Sensing team inoculate LB medium with any transformant cultures which grow on these plates - if there are no cultures present then the transformations will be attempted a second time.''' | ||
| + | |||
| + | ===Observations=== | ||
| + | [[Image:Team Newcastle 2009 iGEM 10-09-09 IMG 1047.JPG|400px|center]] | ||
| + | <br> | ||
| + | On all four plates there could be seen hundreds of transformant colonies - this was to be expected as the DNA used to transform the ''DH5-alpha'' cells was in pure form. | ||
| + | |||
| + | ===Procedure=== | ||
| + | |||
| + | There were two procedures carried out in response to these observations: | ||
| + | <br> | ||
| + | * Three colonies from each 200ul plate of transformants were used to inoculate 6 x 5ml LB+kanamycin (making a total of six tubes of inoculated 5ml LB + kanamycin). This is preparation for mini-preps. | ||
| + | <br> | ||
| + | * One colony from each of the 200ul plates (1 ''cotC-GFP-smtA'' transformed ''E.coli'' colony and 1 ''kinA'' transformed ''E.coli'' colony) was taken to inoculate 2 x 50ml LB + kanamycin media. This is preparation for midi-preps. | ||
| + | <br> | ||
| + | ====Preparations for Mini-preps==== | ||
| + | Three colonies were extracted from the 200ul ''cotC-GFP-smtA'' transformant cells plate and used, under aseptic conditions, to inoculate 3 x 5ml LB+kanamycin tubes. In the same way three colonies from the 200ul ''kinA'' transformant cells plate was used to inoculate 3 x 5ml LB+kanamycin tubes. These six tubes were then placed in the orbital shaking incubator at 37C for overnight growth. | ||
| + | <br> | ||
| + | ====Preparations for Midi-preps==== | ||
| + | |||
| + | When setting up 50ml LB cultures for midi-preps it is usual practice to inoculate the 50ml of LB with 50ul of LB culture taken from an overnight growth. However time was running short so we decided to take a risk and inoculate two flasks of 50ml LB + kanamycin directly from plates; one flask inoculated with 1 colony of ''kinA'' transformants and the second flask of 50ml LB+kanamycin inoculated with 1 colony of ''cotC-GFP-smtA'' transformants. These flasks were too placed in the orbital shaking incubator at 37C for overnight growth. | ||
| + | |||
| + | ==<u>Stochastic Switch Team</u>== | ||
| + | |||
| + | Today we miniprepped sac and ara and carried out a test digest (EcoRI and PstI) on the samples in order to check if the insert hed ligated correctly. We also digested Hanny and James' sleB miniprep which needed to be cut with NheI and EcoRI. | ||
| + | As it happened clear bands could not be seen for the ara and sleB lanes. | ||
| + | |||
| + | ==<u>Sporulation Tuning/Chassis Team</u>== | ||
| + | |||
| + | ===Introduction=== | ||
| + | The transformation of sleB:pSB1AT3 did work with DH5-alpha strain, but no colony grows for JM109 strain. We need pick colonys for mini prep to check whether our transformation was right or not.Here I made a mistake , for I only pick up 1 colony for mini prep. After we got the transformation result, we should pick more colonys for mini prep because we can't assure that all colonys contains right clone. My single colony turn out to be the wrong one. | ||
| + | <br> | ||
| + | |||
| + | ===Futher plan=== | ||
| + | * Mini Prep. for transformation. | ||
| + | * RePCR cwlJ to get our biobrick using original cwlJForward and cwlJReverse primers. | ||
| + | |||
| + | {{:Team:Newcastle/Project/Labwork/CalTemplate}} | ||
{{:Team:Newcastle/Footer}} | {{:Team:Newcastle/Footer}} | ||
{{:Team:Newcastle/Right}} | {{:Team:Newcastle/Right}} | ||
Latest revision as of 12:14, 20 August 2010
Project
Sub-projects
Wet Lab
Meetings
Planning
Links
Formal Lab Session - 10th September 2009
Overview
Metal Sensor Team
Introduction
Yesterday saw the Metal Sensing team attempt to transform DH5-alpha E.coli bacterial cells with two BioBricks (the cotC-GFP-smtA BioBrick and the kinA BioBrick) and plate them out on LB + kanamycin plates. The cotC-GFP-smtA BioBrick is part of the Metal Sensing team's sub-project and the kinA BioBrick will be used by the Stochastic Switch team's sub-project.
Today's exercise should see the Metal Sensing team inoculate LB medium with any transformant cultures which grow on these plates - if there are no cultures present then the transformations will be attempted a second time.
Observations
On all four plates there could be seen hundreds of transformant colonies - this was to be expected as the DNA used to transform the DH5-alpha cells was in pure form.
Procedure
There were two procedures carried out in response to these observations:
- Three colonies from each 200ul plate of transformants were used to inoculate 6 x 5ml LB+kanamycin (making a total of six tubes of inoculated 5ml LB + kanamycin). This is preparation for mini-preps.
- One colony from each of the 200ul plates (1 cotC-GFP-smtA transformed E.coli colony and 1 kinA transformed E.coli colony) was taken to inoculate 2 x 50ml LB + kanamycin media. This is preparation for midi-preps.
Preparations for Mini-preps
Three colonies were extracted from the 200ul cotC-GFP-smtA transformant cells plate and used, under aseptic conditions, to inoculate 3 x 5ml LB+kanamycin tubes. In the same way three colonies from the 200ul kinA transformant cells plate was used to inoculate 3 x 5ml LB+kanamycin tubes. These six tubes were then placed in the orbital shaking incubator at 37C for overnight growth.
Preparations for Midi-preps
When setting up 50ml LB cultures for midi-preps it is usual practice to inoculate the 50ml of LB with 50ul of LB culture taken from an overnight growth. However time was running short so we decided to take a risk and inoculate two flasks of 50ml LB + kanamycin directly from plates; one flask inoculated with 1 colony of kinA transformants and the second flask of 50ml LB+kanamycin inoculated with 1 colony of cotC-GFP-smtA transformants. These flasks were too placed in the orbital shaking incubator at 37C for overnight growth.
Stochastic Switch Team
Today we miniprepped sac and ara and carried out a test digest (EcoRI and PstI) on the samples in order to check if the insert hed ligated correctly. We also digested Hanny and James' sleB miniprep which needed to be cut with NheI and EcoRI. As it happened clear bands could not be seen for the ara and sleB lanes.
Sporulation Tuning/Chassis Team
Introduction
The transformation of sleB:pSB1AT3 did work with DH5-alpha strain, but no colony grows for JM109 strain. We need pick colonys for mini prep to check whether our transformation was right or not.Here I made a mistake , for I only pick up 1 colony for mini prep. After we got the transformation result, we should pick more colonys for mini prep because we can't assure that all colonys contains right clone. My single colony turn out to be the wrong one.
Futher plan
- Mini Prep. for transformation.
- RePCR cwlJ to get our biobrick using original cwlJForward and cwlJReverse primers.
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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)
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]
