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SEPTEMBER: WEEK 1
August, 30th
Colony PCR was performed on <partinfo>BBa_J23116</partinfo>_4C5 (3 colonies) and <partinfo>BBa_J23118</partinfo>_4C5 (3 colonies), since they were negative at the past PCR screening (see August, 27th).
Gel results are shown in the image below. As you can see, all the clones were correct, so we kept the first colony for both and prepared glycerol stocks.
<partinfo>BBa_J23116</partinfo>_4C5 and <partinfo>BBa_J23118</partinfo>_4C5 are stored at -80°C and soon will be tested with the other clones we prepared and screened last week.
 PCR results for <partinfo>BBa_J23116</partinfo>_4C5 1, 2 and 3 and for <partinfo>BBa_J23118</partinfo>_4C5 1, 2, 3. The last lane is reaction blank.
Transformation of ligations I47, I48, I49 into E. coli DH5-alpha. Strains were plated on LB+Amp agar plates and let grow at 37°C.
August, 31st
Glycerol stocks were prepared for
picked yesterday from I29 ligation agar plate.
Remaining culture was MiniPrepped to purify DNA.
| Culture | Quantification
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| I29-1 | 149,6 ng/ul
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| I29-2 | 110,3 ng/ul
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| I29-3 | 91,0 ng/ul
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DNA was digested for 3 hours at 37°C as follows:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 (ul) | Enzyme 2 (ul) | Buffer H (ul)
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| I29-1 | Insert/Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I29-2 | Insert/Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I29-3 | Insert/Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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and than gel run to check the length of ligations.
 Screening for I29-1, I19-2 ans I29-3. All samples are positive; we took I29-1 because its run was cleaner.
Sudan Black staining protocol for BioPlastic:
Sudan Black and Safranine stain were prepared:
- Sudan Black 0,3% in EtOH 70% (0,1 g Sudan Black in 33,3 ml EtOH 70%)
- Safranine 0,5% in H20 (1,25 g Safranine in 250 ml ddH20)
Cultures of PBHR68 and RBS were diluted 1:100 this morning and let grown till they reached an OD600 0,06. When they reached this optical density, they were induced with 1mM IPTG and following samples were prepared:
- RBS (C-)
- PBHR68 with NOTHING added
- PBHR68 with 2% glycerol added
- PBHR68 with 1mM IPTG added
- PBHR68 with 2% glycerol and 1mM IPTG added
After 8 hours, microscope slides were prepared with the usual protocol.
PCR from colony for I47, I48, I49 ligations. Three colonies were picked from each plate, and they were also let grow in LB+Amp to make glycerol stocks of the positive ones.
 Colony PCR for I47, I48, I49 ligations. Only I48-1 and I49-1 are positive, we will try PCR again for I47.
September, 1st
PCR from colony for I47 ligation. Six colonies were picked from the plate, and they were also let grow in LB+Amp to make glycerol stock.
 Colony PCR for I47 ligation. Unfortunately none of them was positive.
Tecan Test was performed on prepared samples, after the usual protocol (dilution, medium change and dilution 1:1000).
September, 2nd
Again PCR from colony for I47 ligation: I47-10..26.
 Colony PCR for I47 ligation: I47-10..26. This time two samples are positive: I47-13 and I47-15. We choose to stock I47-13 as I47.
Trasformation of:
- I44 into DB3.1 (LB+Cm12.5);
- C0083 into DH5-alpha (LB+Kan);
- pSB1C3 (2009 distribution) into DB3.1 (LB+Cm34);
- pSB1C3 (2010 distribution) into DH5-alpha (LB+Cm34)
Tecan Test was performed on prepared samples, after the usual protocol (dilution, medium change and dilution 1:1000).
September, 3rd
Today we got our yeast strains: storage at -80°C.
Inoculum of:
- MG42-1
- MG42-2
- MG42-3
- MG43-1
- MG43-2
- MG43-3
- MC42-1
- MC42-2
- MC42-3
- MC43-1
- MC43-2
- MC43-3
- MC1061 YALE
into 20ul of LB.
PCR of samples inoculated was performed.
Miniprep and digestion of 4C5.
Quantification at Nanodrop: 272.8 ng/ul.
Ligation of I51=MyCrem(E-P)+ccdB(E-P).
PCR of I42-2 was performed.
September, 4th
Results of genomic integration of samples inoculated yesterday:
- LB+Amp50: in LB broth all died, on plate all died (except for J100)
- LB+Cm8:
- MG42-1/2/3 all died
- MG43-1/2/3, MC42-1/2/3, MC43-1/2/3 all muddy
- MC1061 muddy. It's not right because it was the negative control.
Following the results obtained, we perfom a backup on plate.
The followung plates were prepared:
- Cm6 (backup) with MG42-1/2/3,MG43-1/2/3, MC42-1/2/3, MC43-1/2/3 and MC1061;
- Cm12.5 (resistence check) with MG42-1/2/3,MG43-1/2/3, MC42-1/2/3, MC43-1/2/3 and MC1061;
- Cm6 (new colonies);
- Cm12.5 (new colonies check);
- Cm6 (MG and MC wild type);
- Cm12.5 (MG and MC wild type);
September, 5th
The results of yesterday's plates were:
- Cm6 (backup)--> ok!
- Cm12 (check)--> ok!
- Cm6 (new colonies)--> ok!
- Cm12.5 (new colonies check)--> ok!
- Cm6 (MG wt)-->ok!
- Cm12 (MG wt)-->nothing.
- Cm6 (MC wt)-->nothing.
- Cm12 (MC wt)-->nothing.
We pick the following colonies:
- 5 colonies for I51 into 5ml of Cm12.5;
- 5 colonies for I44 into 5ml of Cm12.5;
- 3 colonies for MG43 into 5ml of LB+Cm12.5 and 1ml of LB+Amp50 as chack;
- 3 colonies for MC42 into 5ml of LB+Cm12.5 and 1ml of LB+Amp50 as chack;
- 3 colonies for MC43 into 5ml of LB+Cm12.5 and 1ml of LB+Amp50 as chack;
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