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Team:Calgary/27 May 2009
From 2009.igem.org
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The purpose of today’s experiments is to verify the presence of luxO D47A in the psB1AC3 vector. A restriction digest (RD) with NotI was set up for 2 hours at 37ºC, with two separate colonies of isolated luxOD47A in psB1AC3 (from May 26). A PCR using LuxO F/R primers (gene specific) was set up using platinum Taq Polymerase (Invitrogen, CA). according to the specifications of the manufacturer. Template from colonies 1 and 2 of luxOD47A in psB1AC3 were used. Conditions were as follows: 94ºC for 3 minutes; 36 X (94ºC for 30 seconds; 53ºC for 30 seconds; 72ºC for 2 minutes); 72ºC for 10 minutes; held at 4ºC. The products from the PCR and the RD were then run on a 1% agarose gel (see below). | The purpose of today’s experiments is to verify the presence of luxO D47A in the psB1AC3 vector. A restriction digest (RD) with NotI was set up for 2 hours at 37ºC, with two separate colonies of isolated luxOD47A in psB1AC3 (from May 26). A PCR using LuxO F/R primers (gene specific) was set up using platinum Taq Polymerase (Invitrogen, CA). according to the specifications of the manufacturer. Template from colonies 1 and 2 of luxOD47A in psB1AC3 were used. Conditions were as follows: 94ºC for 3 minutes; 36 X (94ºC for 30 seconds; 53ºC for 30 seconds; 72ºC for 2 minutes); 72ºC for 10 minutes; held at 4ºC. The products from the PCR and the RD were then run on a 1% agarose gel (see below). | ||
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For the RD, we should see two bands for the “cut”: a band at ~3kb for the psB1AC3 vector, and a band at ~1.4kb for luxO D47A. However, the “cut” lanes show that NotI was unable to cut out any insert, suggesting the absence of luxO D47A. However, if we look to the PCR for colony 1 and 2, we do see the expected band size of ~1.4kb for the luxO D47A gene. The band size lines up nicely with the positive control, Lux O D47A in TOPO T/A. However, the negative control lane shows contamination | For the RD, we should see two bands for the “cut”: a band at ~3kb for the psB1AC3 vector, and a band at ~1.4kb for luxO D47A. However, the “cut” lanes show that NotI was unable to cut out any insert, suggesting the absence of luxO D47A. However, if we look to the PCR for colony 1 and 2, we do see the expected band size of ~1.4kb for the luxO D47A gene. The band size lines up nicely with the positive control, Lux O D47A in TOPO T/A. However, the negative control lane shows contamination | ||
Revision as of 00:21, 4 August 2009
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CAROL
Modeling Readings
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CHINMOYEE
Descriptive Title of What You're Doing
WIKI CODING HERE
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EMILY
Restriction Digest and PCR with gene-specific primers of LuxOD47E in the psB1AC3 vector
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FAHD
Descriptive Title of What You're Doing
WIKI CODING HERE
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IMAN
Descriptive Title of What You're Doing
WIKI CODING HERE
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JAMIE
Descriptive Title of What You're Doing
WIKI CODING HERE
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JEREMY
Verifying the presence of luxO D47A in the psB1AC3 vector
The purpose of today’s experiments is to verify the presence of luxO D47A in the psB1AC3 vector. A restriction digest (RD) with NotI was set up for 2 hours at 37ºC, with two separate colonies of isolated luxOD47A in psB1AC3 (from May 26). A PCR using LuxO F/R primers (gene specific) was set up using platinum Taq Polymerase (Invitrogen, CA). according to the specifications of the manufacturer. Template from colonies 1 and 2 of luxOD47A in psB1AC3 were used. Conditions were as follows: 94ºC for 3 minutes; 36 X (94ºC for 30 seconds; 53ºC for 30 seconds; 72ºC for 2 minutes); 72ºC for 10 minutes; held at 4ºC. The products from the PCR and the RD were then run on a 1% agarose gel (see below).
700px For the RD, we should see two bands for the “cut”: a band at ~3kb for the psB1AC3 vector, and a band at ~1.4kb for luxO D47A. However, the “cut” lanes show that NotI was unable to cut out any insert, suggesting the absence of luxO D47A. However, if we look to the PCR for colony 1 and 2, we do see the expected band size of ~1.4kb for the luxO D47A gene. The band size lines up nicely with the positive control, Lux O D47A in TOPO T/A. However, the negative control lane shows contamination
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KATIE
Descriptive Title of What You're Doing
WIKI CODING HERE
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KEVIN
Verification of Pqrr4 for Reporter circuit
Restriction Digest
To verify the isolated Pqrr4, restriction digest was done with NotI (invitrogen, Lot 2803027) enzymes, React Buffer 3, and GeneRuler SM1333 ladder.
Polymerase Chain Reaction (PCR) The Pqrr4 was then amplified using Polymerase Chain Reaction (PCR) from biobrick AC (pSB1AC3) using primers Pqrr4 F/R and platinum Thermus aquaticus (pTaq; invitrogen) according to the specifications of the manufacturer. The following cycling conditions were used: 94°C for 3 min; 36x (94°C for 30s; 53°C for 45s; 72°C for 1 min;) 72°C for 10 min; held at 4°C.
result
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MANDY
Descriptive Title of What You're Doing
WIKI CODING HERE
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PATRICK
Descriptive Title of What You're Doing
WIKI CODING HERE
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PRIMA
Descriptive Title of What You're Doing
WIKI CODING HERE
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STEFAN
Descriptive Title of What You're Doing
WIKI CODING HERE
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VICKI
NotI restriction digest of LuxPQ BBk on psB1AC3
Purpose: Materials and methods: Results:
PCR amplification of LuxPQ BBk
Purpose: Materials and methods: TOPO clones of LuxPQ are used as a positive control. Since the gene of interest (LuxPQ) is purportedly present in these (according to sequencing results from last year), you would expect to see a band at 3855 bp. If the BBk bands are also this size, it encourages our presumption that they are indeed LuxPQ BBk and not some other gene that can’t be amplified by primers specific to LuxPQ. ddHOH is our negative control. Note: the BBk and TOPO LuxPQ are very concentrated. More dilute samples of each were made (100 ng/uL) so that an appropriate volume with lower error could be pitetted into the PCR tubes. 49 uL of master mix was pipetted into each tube, followed by the BBk samples, the TOPO samples and the negative control, according to the following key:
Master Mix contents (for 9 tubes)
TOTAL: 441 uL of Master Mix, of which 49 uL were spread across 8 tubes PCR steps:
Results:
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