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Team:Calgary/22 June 2009
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| - | + | JUNE 22, 2009 | |
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| - | + | Colony PCR and Enzyme Digestion of Mutated Plasmid | |
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| - | + | * Attempted to perform colony PCR and enzyme digestion with XbaI on the mutated plasmid with a positive control (non-mutated plasmid) | |
| + | * Results showed that plasmid does NOT contain the XbaI site (the 0.6% agarose gel showed that there is only one 6KB band from the colonies whereas the positive control showed two bands) | ||
| + | * Will try to isolate higher concentrations for sequencing. | ||
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Looked into Papers : | Looked into Papers : | ||
| - | + | *Imapct of Stochasticity on gene regulatory networks | |
| - | + | *Analysis of Noise in AI-2 Circuits | |
| - | + | *Stochasticity in Transcriptional Regulation: Origins, Consequences,and Mathematical Representations | |
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*Performed a Colony PCR on LuxOD47E in psB1AC3 vector colonies from last week. Only digests done with XbaI / PstI enzymes produced colonies on plates, so these were the only colonies used. | *Performed a Colony PCR on LuxOD47E in psB1AC3 vector colonies from last week. Only digests done with XbaI / PstI enzymes produced colonies on plates, so these were the only colonies used. | ||
*Used p Taq and LuxO forward and reverse primers. Cycling conditions were as follows: 94 C for 6 minutes, 36x (94 C for 30 sec, 55 C for 45 sec, 72 C for 90 sec), 72 C for 10 minutes and hold at 4 C. | *Used p Taq and LuxO forward and reverse primers. Cycling conditions were as follows: 94 C for 6 minutes, 36x (94 C for 30 sec, 55 C for 45 sec, 72 C for 90 sec), 72 C for 10 minutes and hold at 4 C. | ||
| - | *See gel photo below. Lanes 1-6 are LuxOD47E cut with XbaI / PstI, colonies 1-6, Lane 7 is a | + | *See gel photo below. Lanes 1-6 are LuxOD47E cut with XbaI / PstI, colonies 1-6, Lane 7 is a negative control with ddH2O. |
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| + | [[Image:2009.06.22ColonyPCR1.jpg|350px]] | ||
*Results: Nothing was amplified. This was because I used the wrong buffer. Will proceed by re-running the Colony PCR with the CORRECT buffer (10x PCR Buffer- Cl2). | *Results: Nothing was amplified. This was because I used the wrong buffer. Will proceed by re-running the Colony PCR with the CORRECT buffer (10x PCR Buffer- Cl2). | ||
*Performed colony PCR again with the proper PCR Buffer this time. PCR products were visualized on a 1% agarose gel run at 120 V with Generuler 1.0 KB+ DNA Ladder. Lanes 1-7 are LuxOD47E in psB1AC3 cut with XbaI / PstI. Lane 8 is a negative control with only Mastermix + ddH2O. | *Performed colony PCR again with the proper PCR Buffer this time. PCR products were visualized on a 1% agarose gel run at 120 V with Generuler 1.0 KB+ DNA Ladder. Lanes 1-7 are LuxOD47E in psB1AC3 cut with XbaI / PstI. Lane 8 is a negative control with only Mastermix + ddH2O. | ||
*See gel photo below. | *See gel photo below. | ||
| - | *From this gel, it looks like LuxOD47E is in the vector as we see the | + | <Br> |
| + | [[Image:2009.06.23ColonyPCR2.jpg|350px]] | ||
| + | *From this gel, it looks like LuxOD47E is in the vector as we see the appropriate band size of approximately 1.4 KB. | ||
*Prepared overnight cultures of the colonies, will isolate plasmid tomorrow. | *Prepared overnight cultures of the colonies, will isolate plasmid tomorrow. | ||
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| - | + | Construction verification | |
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| - | + | Colony PCR could not be used as a means to verify due to the presence of intrinsic terminators (B0015) within the plasmid backbone. If B0015 gene-specific primers are used, multiple bands will likely appear. Instead a ''Not''I digest coupled with a size control will be used as a verification. Standard manufacturer's specifications followed. | |
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| - | + | PCR of luxPQ in psB1AK3 using BBK-CP-F/R and gene specific primers | |
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| - | + | Purpose: to verify presence of LuxPQ in psB1AK3. Colony 4 of luxPQ in psB1AK3 was used as a template and pTaq DNA polymerase was used. LuxPQ-F/R gene specific primers and BBK-CP-F/R primers (that anneal just outside the BBK prefix and suffix) were used. Conditions were as follows: 94ºC for 2 minutes; 36X (94ºC for 30 seconds; 55ºC for 45 seconds for BBK-CP-F/R primers (53ºC for 45 seconds for LuxPQ F/R primers); 72ºC for 4 minutes); 72ºC for 10 minutes; held at 4ºC. The products were then run on a 0.8% agarose gel (see below). Again, we are looking for a band size of just under 4kb, which we do not see here, and we must thus restart from luxPQ in TOPO. | |
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| - | + | [[Image:2009.06.22.LuxPQ_BBKCP_BBK.png|700px]] | |
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Restreak of R0040, B0015, and J13002 were done to grow more of verified single colonies. | Restreak of R0040, B0015, and J13002 were done to grow more of verified single colonies. | ||
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I learned the purpose and procedures for restriction digest from shadowing Jeremy. After hanging out with in the lab for some time, I began understanding what PCR was used for, when PCR needed to be done, what's the water bath for and much more. | I learned the purpose and procedures for restriction digest from shadowing Jeremy. After hanging out with in the lab for some time, I began understanding what PCR was used for, when PCR needed to be done, what's the water bath for and much more. | ||
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Latest revision as of 06:25, 20 October 2009
UNIVERSITY OF CALGARY
