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Things we did today

Wetlab work

Test of lab's RE (cont.)

EtBr stained 1.2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of RE digest reaction were loaded into each well.

Grace

• Ran RE digests from yesterday on a 1.2% agarose gel

• BamHI and HindIII digested DNA not visualized. Too little DNA added?
• NotI works. All plasmid preps except I14032 resulted in 2 bands of the correct size.

• Note: There is genomic DNA in all preps (band that looks like fire up top)

Verified transformants

Grace

EtBr stained 2% agarose gel ran at 95V for 1 hour. Five microliters of PCR reaction were loaded into each well.

• Colony PCR of transformants to check for correct insert

• GFP+tt = unsuccessful
• GFPf+tt colonies 1, 3, 5 good
• slr1+GFPf colony 5 potentially good
• pilA+GFPf colonies 1, 2, 3, 4 potentially good

• Ligated slr, pilA with GFPf (ES digested) instead of GFPf (XP digested). Still got colonies, some which are potentially good. Hm...

• Restreaked:

• GFPf+tt colony 5
• slr1+GFPf colony 5
• pilA+GFPf colonies 1 and 4

• Will colony PCR again tomorrow to prep for sequencing

3A assembly

Grace

• Ligated p+r with g or s onto pSB1A2

• pnir+rbs with GFP, slr1, pilA
• plac+rbs with GFP, slr, pilA

• Transformed into DH5α using 5 μl ligation reaction

Rear ligation of signal sequences and GFPf

Grace

• Ligated slr and pilA with XbaI and PstI digested GFPf
• Transformed into DH5α using 5 μl ligation reaction

[[Editing Team:Hawaii/Notebook/2008-08-11|Transformation

Margaret

• Re-streaked yesterday's transformation
• Colony PCR on each of the re-streaked colonies

Construction of Omega Interposon

Margaret

• Digested pSMC121 and pSB1A2 with SmaI
• Ligated parts together (over-night ligation)

Plasmid Prep

Margaret

• B0015, B0030, E0040, J33207
• Drying in hood

Glycerol Stock

Margaret

• oriT (3 bottles) and I14032 (1 bottle)

Media Making

Margaret

• Amp100 plates, Solution 3 for plasmid preps, 100mM IPTG(look in -20C with the antibiotics)

Drylab Work

Sequencing

Grace

• Downloaded sequences from CORE Hawaii. Began assembling contigs and checking sequences.

Discussion

Quote of the Day

History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson

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