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Things we did today

Wetlab work

Construction of p+r

EtBr stained 2% agarose gel ran at 60V for 2.25 hours. Thirty microliters of RE digested DNA were loaded into each well.

Grace

• Ran RE digested products on gel

• nir had three bands. Band at ~730bp = ?
• BB-pRL1383a -- still couldn't see cut out of GFP. Assumed double digest successful, too little DNA to see GFP.
• J33207 wrong size

• Extracted nir, C0012 vector, BB-pRL1383a from gel
• 3A assembly (ligation) of C0012 vector (pSB1A2) with:

• nir and rbs (B0034)
• plac and rbs(B0034)
• pSB1A2 (negative control)

• Transformed DB3.1 cells using 5 μl ligation reaction

Transformation

Margaret

• ligation products from yesterday were transformed into DH5-a cells, pSMC121 was used as a positive control
• rep+pSB1A3 (1:1)
• rep+pSB1A3 (1:3)
• rep+pSB1A3 (1:6)
• P1 lytic +pSB1A3 (1:1)
• P1 lytic +pSB1A3 (1:3)
• P1 lytic +pSB1A3 (1:6)
• [Plac B0030]+pSB1A3 (1:3)
• [Plac B0030]+pSB1A3 (1:6)
• [Plac B0034]+pSB1A3 (1:6)**ran out of vector

Sequencing

Gel of the pcr in preparation for sequencing, also colony pcr of rep and lytic regions.

Margaret

• in preparation for sequencing, a PCR was run on the following plasmids:

oriV1-4, aadA(BB)5, aadA(BB)9, aadA(pRL)3, aadA(pRL)6, omega interposon

• Note: In lane five, it looks like I added oriV instead of aada(BB)5. In the omega lane, there is a ladder. I will still sequence these.

Grace

• Prepared nir+rbs and rbs+GFP constructs for sequencing

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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