Latest revision as of 19:16, 15 August 2008

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Things we did today

Wetlab work

Colony PCR

PCR verification of aadA from pRL1383a using wrong conditions.

File:Rep P1 omega colony pcr.jpg

PCR verification of rep, P1 lytic regbion, and omega interposon ligation from 8-13

Margaret

5 colonies of rep, P1 lytic region, and aadA(pRL1383a) region, each. pSB1A3 was used as a control.
I changed the annealing time to 3min 30 seconds instead of the extension time, so the sites were not amplified properly. I ran the aadA(pRL1383a) construct on a gel and got a few bands, none of which were the right size. One of them should have been right though, so I am assuming this ligation did not work.
I ran another PCR verification on rep, P1, and the omega interposon and afterwards a gel.

Krystle

Krystle update

Culture

Margaret

Plasmid preps from yesterday did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.

Krystle

Inoculated for plasmid prep of GFPf and J04430 tomorrow.

Restriction Digest

Margaret

digest of I14032 with SpeI and PstI. I want to use this to ligate to the aadA(BB) construct.

Grace

Checked Krystle's RE digests from last night on a gel

Also ran J33207 PCR product on same gel
Excised J33207 and GFPf bands from gel

RE digested:

EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digests were loaded into each well. BB-pRL1383a was digested with EcoRI/PstI. Picture is mislabeled.

J33207, J04430, BB-pRL1383a with EcoRI and PstI
pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
BB-pRL1383a with HindIII and BamHI (from lab -20C)

Ran RE digests on a gel to purify segments of interest

Also ran RE digested B0015 from Krystle

Gel extraction

Krystle

Used Epoch Biolabs kit to extract J33207 and GFPf DNA from agarose gel

Drylab Work

Sequencing

Margaret

Verified sequence of oriT, inserted into pSB1A2. The sequences of oriT and the BioBrick sites are correct.

Oligonucleotide Design

Margaret & Grace

We are having trouble with TT, RBS and Promoter ligations, so we decided to synthesize these parts.
B0016, I14032, B0030, & B0034

Discussion

Gel box 1 is the only one that works properly. The unlabeled gel box and box #2 may cause gels to run excessively slow, bands to blur/smear, or other weird things. Amps is really low on these boxes. All covers/power sources are good.

Quote of the Day

Happy chickens taste better. - KSGSK

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

@@ Line 4: / Line 4: @@
 == Wetlab work ==
 ===Colony PCR===
+[[Image: aada_verification_8_14.jpg|right|thumb|150px|PCR verification of aadA from pRL1383a using wrong conditions.]][[Image:rep_P1_omega_colony_pcr.jpg|right|thumb|150px|PCR verification of rep, P1 lytic regbion, and omega interposon ligation from 8-13]]
 :<strong> Margaret </strong>
 :* 5 colonies of rep, P1 lytic region, and aadA(pRL1383a) region, each. pSB1A3 was used as a control.
-[[Image: aada_verification_8_14.jpg|right|thumb|300px|PCR verification of aadA from pRL1383a using wrong conditions.]]
 :* I changed the annealing time to 3min 30 seconds instead of the extension time, so the sites were not amplified properly. I ran the aadA(pRL1383a) construct on a gel and got a few bands, none of which were the right size. One of them should have been right though, so I am assuming this ligation did not work.
 :*I ran another PCR verification on rep, P1, and the omega interposon and afterwards a gel.
-[[Image:rep_P1_omega_colony_pcr.jpg|right|thumb|300px|PCR verification of rep, P1 lytic regbion, and omega interposon ligation from 8-13]]
+:<strong>Krystle</strong>
+:* '''Krystle update'''
 ===Culture===
-<strong>Margaret</strong>
+:<strong>Margaret</strong>
-:*Plasmid preps from [[Team:Hawaii/Notebook/2008-08-14|yesterday]] did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.
+:*Plasmid preps from [[Team:Hawaii/Notebook/2008-08-14 | yesterday]] did not yield very high quantities. I am setting up the culture for oriV1-4 and aadA(BB) 4 so I can do another plasmid prep tomorrow.
+:<strong>Krystle</strong>
+:* Inoculated for plasmid prep of GFPf and J04430 tomorrow.
 ===Restriction Digest===
+:<strong>Margaret</strong>
-<strong>Margaret</strong>
 :*digest of I14032 with SpeI and PstI. I want to use this to ligate to the aadA(BB) construct.
+:<strong>Grace</strong>
+:* Checked Krystle's RE digests from last night on a gel
+::* Also ran J33207 PCR product on same gel
+::* Excised J33207 and GFPf bands from gel
+:* RE digested: [[Image:081408REdigests.jpg|right|thumb|200px|EtBr stained 2% agarose gel ran at 72V for 1.5 hours. Thirty microliters of the RE digests were loaded into each well. BB-pRL1383a was digested with EcoRI/PstI. Picture is mislabeled.]]
+::* J33207, J04430, BB-pRL1383a with EcoRI and PstI
+::* pRL1383a, J33207 with HindIII and BamHI (Roche, from SC)
+::* BB-pRL1383a with HindIII and BamHI (from lab -20C)
+:* Ran RE digests on a gel to purify segments of interest
+::* Also ran RE digested B0015 from Krystle
+===Gel extraction===
+:<strong>Krystle</strong>
+:* Used Epoch Biolabs kit to extract J33207 and GFPf DNA from agarose gel
 == Drylab Work ==
@@ Line 36: / Line 54: @@
 = Discussion =
+:* Gel box 1 is the only one that works properly. The unlabeled gel box and box #2 may cause gels to run excessively slow, bands to blur/smear, or other weird things. Amps is really low on these boxes. All covers/power sources are good.
 = Quote of the Day =
-<blockquote>''History is the only laboratory we have in which to test the consequences of thought.'' - Étienne Gilson</blockquote>
+<blockquote>''Happy chickens taste better.'' - KSGSK</blockquote>
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