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Team:Bordeaux/Second
From 2012.igem.org
Day 5 : 09-07-2012
Today we didn't get our new competent cells stock so we decided to try again with the plates from last week but with other colonies. We inoculated two 5 ml LB + Ampicilin 100μg/mL tubes per plate and put them to incubate at 30°C for 19h.
Day 6 : 10-07-2012
Every tube grown well overnight except for the two 1.B tubes wich are clear after centrifugation we confirmed that nothing grew on the two 1.B tubes. We did a miniprep for the other tubes , As usual we used a QIAprep® Spin Miniprep kit from QIAGEN® Read Protocol We checked the samples concentration with the nanodrop Read Procedure We still got bad results ( around 15ng/μL) except for one 1.D tube ( 38,3ng/μL) and one 5.B tube ( 28ng/μL) We decided to run an agarose 1% gel electrophoresis to confirm what we got on the samples
1.A = BBa_I14015 + pSB2K3 = 4.6 kb 1.D = BBa_C0051 + pSB1A2 = 2.8 kb 5.B = BBa_B0015 + pSB1AK3 = 3.3 kb We can see that the samples contain the good plasmids The strange thing is that the concentration seems higher in the 1.A and 5.B samples
We got one sample of XL1blue strain competent cells to test So we did an electroporation as usual Read Protocol and took 1.B DNA to transform We plated 50μL and 100μL the transformants on two LB + Ampicilin 100μg/mL plates We then put them to incubate at 37°C overnight
Day 7 : 11-07-2012
Today we received the package from our team sponsor Thermo scientific We got the Fastdigest® restriction enzymes we ordered. We also got a 250 GeneJET™ Plasmid Miniprep Kit Read Protocol The plates put to grow overnight gave poor results no more than 10 colonies in each plate We decided to try to regrow the transformants put to freeze on day 3 and to do the low copy plasmids miniprep procedure on the cultures.
We put them and Two XL1 blue 1.B colonies on two 5ml LB + Ampicilin 100μg/mL except for 1.A in LB + Kanamicin 50μg/mL The tubes were put to incubate 16h at 37°C
Day 8 : 12-07-2012
Every tube grown well overnight except for the two 1.E tubes wich are clear after centrifugation we confirmed that nothing grew on the two 1.E tubes We did a miniprep for the other tubes , We used our new GeneJET™ Plasmid Miniprep Kit Read Protocol We checked the samples concentration with the nanodrop Read Procedure.
We got better results than with the QIAGEN® kit We decided to run an agarose 1% gel electrophoresis to confirm what we got on the samples
Expected sizes : 1.A = 4.6 kb 1.B = 2.1 kb 1.C = 2.6 kb 1.D = 2.8 kb 5.A = 2.1 kb 5.B = 3.3 kb On this gel we can see that there is a purity problem on 1.C sample and that 1.B samples are out of place , near the 3.5 kb marker tommorow we will run a gel after restriction to identify the problem with 1.B and 1.C samples.
Day 9 : 13-07-2012
Today we tried to identify the biobricks we had on 1.B , 1.B xl1bue and 1.C samples We proceeded by doing a restriction with EcoR1 and PST1 in order to extract the biobrick from the plasmid We the run a gel to identify the plasmid and the biobrick
The gel confirm that 1.C sample is contaminated by another plasmid as we can identify two biobricks ≈ 0.6 kb and ≈ 0.75 kb The sample may be contaminated by 1.D For 1.B samples we can identify the pSB1A2 plasmid at ≈ 2 kb But we can't see the biorbrick extracted as it may be only 56 bp long We ran a 1.5 % agarose gel with a 100 pb ladde
The absence of DNA near the 50 bp marker means that there is not the BBa_K091110 biobrick present in the sample Next week we will replace it by BBa_K091111
