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Things we did today

Wetlab work

Restreaked nir+rbs and I14032+rbs constructs

Grace

Plasmid prep (finished up)

Grace

• Resuspended plasmid preps in 50 μl TE buffer
• Determined DNA concentrations of plasmids

Prep for sequencing

EtBr stained 4% agarose gel ran at 60V for 100 min. Twenty-five microliters of the PCR reactions were loaded into each well.

Grace

• 25 μl PCR reactions of nir+rbs, I14032+rbs, slr1, slr2, BB-pRL1383a

• Colony PCRs seem to indicate success

• 25 μl PCR reactions of GFP+tt, GFPf+tt, J33207+tt

• Colony PCR indicates no ligation. Picked new colony, sequence to confirm failure.

• PCR of nir, B0015, B0030, B0034 for resequencing (bad reads last time)
• Gel purified all PCR rxns (we still have a problem with contaminant DNA/shadow bands) and desired bands were extracted from gel

• 2% agarose gel ran at 60v for 2 hours

• Determined DNA concentrations via nanodrop spectrometer
• Prepared samples and sent to CORE Hawaii for sequencing

Construct p+r+g and p+r+s

Krystle

• Restriction Digest
  • nir+B0030, I14032+B0030, J33207 digested with SpeI and PstI
  • gfp, gfpfusion, and B0015 digested with XbaI and PstI
• Gel Purified restriction digest
  • 2% agarose gel ran at 60 volts for 1.5 hours

40ul of the total restriction digest loaded into each well

• gfpfusion cut out from gel

Discussion

Quote of the Day

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

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

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