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CAROL
Colony PCR
- Only one colony was present on plates and continued with colony PCR using forward and reverse gene specific primers.
- Results showed that colony did not have plasmid because no bands appeared on gel.
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EMILY
Visualization of Colony PCR
- Ran colony PCR Product from yesterday on a 1% agarose gel with 1.0kb Generuler DNA Ladder. See gel below. Lane 1 is BBK LuxOD47E cut with EcoRI/PstI Colony 4, lane 2 is BBK LuxOD47E cut with XbaI/PstI, lane 3 is LuxOD47E C4-8 TOPO DNA, lane 4 is a negative control, lane 5 is BBK LuxOD47E cut with EcoRI/PstI Colony 4, lane 6 is BBK LuxOD47E cut with XbaI/PstI, Colony 2, lane 7 is a positive control with only the psB1AC3 vector and lane 8 is a second negative control.
- Analysis: The negative control lanes are clean, which is a very good sign as this indicates that we have no contamination. All of the bands are also at the expected sizes. The first three lanes show (faint) bands around 1.4 kb, which is expected as this is approximately the size of trhe gene of interest (1362 bp). The fifth lane shows bands.
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JEREMY
Repeat restriction digest of LuxPQ BBK PCR product and psB1AC3
Purpose: To effectively digest the LuxPQ insert and psB1AC3 vector overnight in order to standardize the genetic part for future construction. The following sets of restriction enzymes were used for overnight digest: XbaI/PstI and EcoRI/PstI. The water bath was set at 37ºC
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KEVIN
Other hyperactive antifreeze proteins
- High Arctic plant rhizosphere(P. putida)
- Mid-gut of frozen beetle larvae (R. erythropolis)
- Frozen/chilled pork sausages (M. cryophilus)
- Antarctic soil (Moraxella sp and P. fluorescens).
None were from bacteria until MpAFP was found
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MANDY
Working on Wiki for Canada Day
The images used in the wiki have been removed as they were hotlinked from other websites. Instead, they have been replaced by those hosted on our photobucket account (still need to figure how much can be uploaded on this wiki here, so just in case, they will be hosted externally for now at least). The menu for the wiki has been updated. I put in a header and transparency coding. I'm learning overlays and positioning to make it look really 'cool'. Eventually the background image of the menu can be changed using scripting which I think will be neat.
In addition to the menu, image sizing is fixed and an RSS feed for our blog has been added to the wiki. This only shows the last 10 posts, so I will have to convert blog updates to their respective sections so that they remain permanently accessible on this site.
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PRIMA
Working on Canada Day
I replied back to company emails and tried my best to answer their questions about our project. I researched a few old companies who had declined our proposals and searched new contacts in different departs. I will call them tomorrow.
In the lab
Shadowed Vicky with Colony PCR of J13002 + JuxOD47A BBk + B0015 on pSB1AK3
Purpose: The bacterial culture growth is prolific. This will verify if the proper gene constructs are included within.
Materials an methods:
- Primers: BBk forward and reverse construction primers
- Master mix: Use pTaq DNA polymerase. The contents will suffice for 15 tubes, and were prepared in the same fashion as on June 26.
- PCR conditions: These are also the same as they were on June 26
Tubes 1/6 were colony 1
tubes 2/7 were col 2
tubes 3/8 were col 3
tubes 4/9 were col 4
tubes 5/10 were col 5
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VICKI
Colony PCR of J13002 + JuxOD47A BBk + B0015 on pSB1AK3 that I prepared yesterday
Purpose: The bacterial culture growth is prolific. This will verify if the proper gene constructs are included within.
Materials an methods:
- Primers: BBk forward and reverse construction primers
- Master mix: Use pTaq DNA polymerase. The contents will suffice for 15 tubes, and were prepared in the same fashion as on June 26.
- PCR conditions: These are also the same as they were on June 26
Results:
The 1% agarose gel is attached below. Lane 1 is a GeneElute 1kb+ DNA ladder; lanes 2-11 are the colony-PCR'd samples of J13002 + LuxOD47A + B0015; lane 12 is a sequenced LuxOD47A + B0015 for a size control; lane 13 is a sequenced LuxOD47A BBk for another size control; and lane 14 is a negative control. Based on a very confusing negative control, this procedure will be repeated tomorrow. We will still progress with a NotI restriction digest of the contents, which will also be done tomorrow.
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