Difference between revisions of "Team:Uppsala/Notebook"

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<h3>Lambda red integration:</h3>
 
<h3>Lambda red integration:</h3>
  
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- PCR of insert CrtEB. Restreak clones of IS150 strain from 21/7 on sucrose and tetracycline plates and gsp strain from 21/7 on and tetracycline and trimetoprim plates. Liquid cultures with same respective antibiotics for the strains were made.<br>
 
- PCR of insert CrtEB. Restreak clones of IS150 strain from 21/7 on sucrose and tetracycline plates and gsp strain from 21/7 on and tetracycline and trimetoprim plates. Liquid cultures with same respective antibiotics for the strains were made.<br>
 
- Gel electrophoresis of CrtEB PCR product from 24/7 (see figure 4). Restreak colonies of IS150 strain from 24/7 plates on sucrose plates then chloramphenicol plates. Prepare cultures for transduction of IS150 by growing overnight in P1 LB. Purify CrtZY and CrtEB and use nano drop on all purified inserts to check DNA concentration (see table 1). Ordered new primers for CrtEB.<br>
 
- Gel electrophoresis of CrtEB PCR product from 24/7 (see figure 4). Restreak colonies of IS150 strain from 24/7 plates on sucrose plates then chloramphenicol plates. Prepare cultures for transduction of IS150 by growing overnight in P1 LB. Purify CrtZY and CrtEB and use nano drop on all purified inserts to check DNA concentration (see table 1). Ordered new primers for CrtEB.<br>
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- gsp strain did not grow on plates but did in liquid culture. 5 of 8 colonies of IS150 strain from 25/7 grew on sucrose but not chloramphenicol (see figure 5 & 6). New plates were made (tet + chlor, tet + tmp, tet + suc, chlor). Transduction of bgl strain as the donor and IS150 as receiver. Take some gsp liquid culture and put in some tet + sucrose to make sure it’s our strain that’s growing. Make liquid cultures with gsp (dhfr) in tet + tmp with the different tet to check if antibiotics work.<br>
 
- gsp strain did not grow on plates but did in liquid culture. 5 of 8 colonies of IS150 strain from 25/7 grew on sucrose but not chloramphenicol (see figure 5 & 6). New plates were made (tet + chlor, tet + tmp, tet + suc, chlor). Transduction of bgl strain as the donor and IS150 as receiver. Take some gsp liquid culture and put in some tet + sucrose to make sure it’s our strain that’s growing. Make liquid cultures with gsp (dhfr) in tet + tmp with the different tet to check if antibiotics work.<br>
 
- No growth on transducer IS150 from 26/7. Start overnight culture of bgl strain to make lysate (donor strain). Colony PCR of the colonies from 26/7 that grew on sucrose but not on chlor and gel electrophoresis to check inserts. Make tet + chlor and tet + tmp plates. Start overnight cultures of IS150 strains with inserts that were confirmed by colony PCR.<br>
 
- No growth on transducer IS150 from 26/7. Start overnight culture of bgl strain to make lysate (donor strain). Colony PCR of the colonies from 26/7 that grew on sucrose but not on chlor and gel electrophoresis to check inserts. Make tet + chlor and tet + tmp plates. Start overnight cultures of IS150 strains with inserts that were confirmed by colony PCR.<br>

Revision as of 15:17, 31 October 2017