Latest revision as of 02:58, 2 August 2008

Birds Eye View Team	Projects	Events	Resources
Sponsors	Experiments	Milestones	Protocols
	Notebook (t)	Meetings (t)

Things we did today

Wetlab work

Colony PCR

Colony PCR: verified plasmids pSB1A3, pSB1A2, pSB3K3, and additionally verifying a sample of pSMC121 graciously donated by Dr.C.

Margaret

Colony PCR of pSB1A2, pSB1A3, pSB3K3

Results

Lane 2 contains bands that are approximately 1kb, while I would expect pSB1A3 to be 991bp. Lane 3 containing pSB1A2 should be 913bp and is just above the 0.9kb mark. Lane 4 contains pSB3K3 and should be 991bp and is just below 1kb.
The positive control, UPA amplifying algal DNA is at 0.5kb as expected.
The negative control, UPA amplifying water, contains no band.
In lane 7 pSMC121, a plasmid containing the omega interposon was run, but no DNA was stained.

Conclusions

The plasmids (pSB1A3, pSB1A2, pSB3K3) were verified using VF2 and VR. I will make a glycerol stock and do a plasmid prep.

The absence of a a band for pSMC121 may explain the difficulty I have had in amplifying the omega region of this plasmid. I need to go get a new sample.

PCR amplification of pRL1383a parts

continued from Saturday, ran a gel

Restriction Digest

Krystle

Performed 2 restriction digests on nir, gfp, and gfp fusion brick with EcoRI and SpeI

trial 1: digested 5ul of each plasmid prep in a 50ul reaction mixture, ran 20 ul of the digest after 3 hours

trial 2: digested 25ul of each plasmid prep in a 50ul reaction mixture, ran 20 ul of the digest after 2 hours

Drylab Work

Name of Task

Krystle, Margaret

We figured out why some of the biobrick extractions did not transform. Some of these parts are on pSB2K3http://partsregistry.org/wiki/index.php?title=Part:pSB2K3 which is a low copy until IPTG is added to the medium in which case it becomes high copy! So next time we make this, add 1mM IPTG to the media.

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 ==
 ===Colony PCR===
+[[Image:Colony_PCR_7_21.jpg|right|thumb|300px|Colony PCR: verified plasmids pSB1A3, pSB1A2, pSB3K3, and additionally verifying a sample of pSMC121 graciously donated by Dr.C.]]
 :<strong>Margaret</strong>
 :* Colony PCR of pSB1A2, pSB1A3, pSB3K3
-[[Image:7_21_08_annotated.jpg|right|thumb|300px|Lane 1 contains the 2-log ladder, lane 2: pSB1A3's ccdB insert, lane3: pSB1A2's ccdB insert, lane 4: pSB3K3's ccdB insert, lane 5: positive control, lane 6: negative control, lane 7: pSMC121.]]
+<strong>Results</strong>
+:*Lane 2 contains bands that are approximately 1kb, while I would expect pSB1A3 to be 991bp. Lane 3 containing pSB1A2 should be 913bp and is just above the 0.9kb mark. Lane 4 contains pSB3K3 and should be 991bp and is just below 1kb.
+:*The positive control, UPA amplifying algal DNA is at 0.5kb as expected.
+:*The negative control, UPA amplifying water, contains no band.
+:*In lane 7 pSMC121, a plasmid containing the omega interposon was run, but no DNA was stained.
+<strong> Conclusions</strong>
+:*The plasmids (pSB1A3, pSB1A2, pSB3K3) were verified using VF2 and VR. I will make a glycerol stock and do a plasmid prep.
+:*The absence of a a band for pSMC121 may explain the difficulty I have had in amplifying the omega region of this plasmid. I need to go get a new sample.
 ===PCR amplification of pRL1383a parts===
@@ Line 25: / Line 38: @@
 ===Name of Task===
-:<strong> name of person/people who performed the task</strong>
+:<strong> Krystle, Margaret</strong>
-:* Summary of task and what was done. Link to experiment for detailed notes if necessary.
+:* We figured out why some of the biobrick extractions did not transform. Some of these parts are on pSB2K3[[http://partsregistry.org/wiki/index.php?title=Part:pSB2K3]] which is a low copy until IPTG is added to the medium in which case it becomes high copy! So next time we make this, add 1mM IPTG to the media.
-:* e.g. read through papers, worked on proposal, etc.

Team:Hawaii/Notebook/2008-07-21

From 2008.igem.org