Latest revision as of 08:28, 28 August 2008

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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 ]

@@ Line 4: / Line 4: @@
 == Wetlab work ==
 ===Construction of p+r===
+[[Image:082708REdigests.jpg|right|thumb|250px|EtBr stained 2% agarose gel ran at 60V for 2.25 hours. Thirty microliters of RE digested DNA were loaded into each well.]]
 :<strong>Grace</strong>
 :* 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:
@@ Line 14: / Line 18: @@
 :* Transformed DB3.1 cells using 5 &mu;l ligation reaction
+===Transformation===
+:<strong>Margaret</strong>
+:* 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===
+[[Image:sequencing_gel.jpg|right|thumb|150px|Gel of the pcr in preparation for sequencing, also colony pcr of rep and lytic regions.]]
+:<strong>Margaret</strong>
+:*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.
+:<strong>Grace</strong>
+:* Prepared nir+rbs and rbs+GFP constructs for sequencing
 = Discussion =

Team:Hawaii/Notebook/2008-08-27

From 2008.igem.org