Imperial College/7 September 2008
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+ | ==Dry Lab: Motility Analysis== | ||
+ | =====Monday===== | ||
+ | *Track 20 cells | ||
+ | *Determine the segmentation of the data: | ||
+ | **plotting velocity to exp(i*theta) | ||
+ | **where theta is the difference between the angle of rotation of the cell's tip to its centroid and the angle of the centroid displacement | ||
+ | |||
+ | =====Tuesday===== | ||
+ | *Track 20 cells | ||
+ | *Prepare for meeting with supervisors | ||
+ | |||
+ | =====Wednesday===== | ||
+ | *Acquire longer microscope videos of swimming B. Subtilis | ||
+ | *Track at least 20 cells manually | ||
+ | *Adjust matlab code according to the segmentation of the data | ||
+ | |||
+ | =====Thursday===== | ||
+ | |||
+ | *Hopefully extract run_time, tumble_time,run velocity and tumble angle using our matlab code | ||
+ | *Plot their distributions and compare to model | ||
+ | |||
+ | =====Friday===== | ||
+ | *Acquire more data by tracking cells manually | ||
+ | *In theory we would need to track about 100 cells to 150 cells | ||
+ | |||
==Wetlab== | ==Wetlab== | ||
+ | ===''B.subtilis''=== | ||
+ | =====Monday===== | ||
+ | *Prepare Lb agar plates and LB media for the week ahead. | ||
+ | *Check PCR from friday on a 1% agarose gel. | ||
+ | *Innoculate 20ml of LB agar for overnight culture | ||
+ | =====Tuesday===== | ||
+ | *Carry out the final protocol for the O.D.<sub>600</sub> vs cell number. From previous trials it is clear that it is better to base the data collection on the O.D.<sub>600</sub> as oppose to regular time intervals. In addition it is clear a greater spread of dilutions are required for an increased data collection. | ||
+ | *Innoculate 20ml of LB agar for overnight culture | ||
+ | |||
+ | =====Wednesday===== | ||
+ | *Microscope from 11-2 to collect wild-type movement of ''B.subtilis'' | ||
+ | *Count the cells from the O.D.<sub>600</sub> vs cell number experiment. | ||
+ | |||
+ | =====Thursday===== | ||
+ | *Innoculate 20ml of LB agar for overnight culture | ||
+ | |||
+ | =====Friday===== | ||
+ | *Microscope from 11-2 to collect wild-type movement of ''B.subtilis'' | ||
===Cloning=== | ===Cloning=== | ||
+ | |||
+ | =====Ongoing/Where there is spare time===== | ||
+ | |||
+ | *PCR trial all genomic sequences to determine optimal conditions with Taq then produce clones with Pfu | ||
+ | *Digest with ''XbaI'' and ''SpeI'' and incorporate into biobricks | ||
+ | *Follow up by assaying orientation (either with sequence internal restriction site or by using ''XbaI'' and ''SpeI'', as they should only be able to cut inserts in the correct orientation) | ||
=====Monday===== | =====Monday===== | ||
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*GeneArt constructs 3, 6, 10 and 12 (EpsE gene and PgsiB-RBSspoVG in construct 3, LipA-Elastin and PgsiB-RBSgsiB in construct 6, P43-RBSspoVG and Pveg-RBSspoVG), along with all above biobricks will be transformed into Xl1-Blue ''E.coli'' | *GeneArt constructs 3, 6, 10 and 12 (EpsE gene and PgsiB-RBSspoVG in construct 3, LipA-Elastin and PgsiB-RBSgsiB in construct 6, P43-RBSspoVG and Pveg-RBSspoVG), along with all above biobricks will be transformed into Xl1-Blue ''E.coli'' | ||
*Depending on results, LacI PCR to be digested and purified ready for ligation into pSB1A2/pSB1AK3 | *Depending on results, LacI PCR to be digested and purified ready for ligation into pSB1A2/pSB1AK3 | ||
- | + | ||
=====Tuesday===== | =====Tuesday===== | ||
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*Constructs 10 and 12 to be digested and gel-purified ready to be incorporated into biobricks | *Constructs 10 and 12 to be digested and gel-purified ready to be incorporated into biobricks | ||
*''E.coli'' containing constructs 3 and 6 to be grown up overnight for midi-prepping | *''E.coli'' containing constructs 3 and 6 to be grown up overnight for midi-prepping | ||
+ | *Mini-prep the LacI biobrick and check for correct orientation. Grow a culture with the LacI gene in the correct orientation for midi-prep tomorrow | ||
*Time permitting (If not may be carried out Friday): | *Time permitting (If not may be carried out Friday): | ||
**Aad9 to be ligated to the P43-RBSgsiB and Pveg-RBSgsiB separately and transformed into XL1-Blue''E.coli'' | **Aad9 to be ligated to the P43-RBSgsiB and Pveg-RBSgsiB separately and transformed into XL1-Blue''E.coli'' | ||
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*Constructs 3 and 6 to be midiprepped | *Constructs 3 and 6 to be midiprepped | ||
*Constructs 3 and 6 digested and run on a gel to separate components by gel purification | *Constructs 3 and 6 digested and run on a gel to separate components by gel purification | ||
+ | *Midi-prep LacI | ||
+ | *Finish any unfinished jobs from earlier in week | ||
+ | |||
+ | <br> | ||
+ | {{Imperial/EndPage|Notebook|Notebook}} |
Latest revision as of 20:45, 28 October 2008
7 September 2008Dry Lab: Motility AnalysisMonday
Tuesday
Wednesday
Thursday
Friday
WetlabB.subtilisMonday
Tuesday
Wednesday
Thursday
Friday
CloningOngoing/Where there is spare time
Monday
Tuesday
Wednesday
Thursday
Friday
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