Tag Archives: COX2

Sequencing – PGS Hi 4 (PGS2/COX2)

Sent plasmid prep to ASU (5uL of plasmid + 1uL of 10uM M13F/R). SJW01 = M13F, SJW02 = M13R.

Results:

Sequencing looks great! Definitely have a portion of the second isoform of COX/PGS!! Here’s the result of the consensus BLASTed in GenBank>Nucleotide (others)>blastn:

Top hit in the db is COX1/PGS1, and, clearly, there are differences between the two sequences confirming that we have the second isoform (COX2/PGS2). Will design more RACE primers in hopes of obtaining the full-length cDNA sequence.

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PCR – Colony PCR on Restreaked PGS2 Clones from 20110707

Ran a colony PCR at the same time that I inoculated liquid cultures, using M13 primers.

Cycling params:

  • 94C – 10m

40 cycles of:

  • 94C – 1m
  • 50C – 1m
  • 72C – 2m

Results:

Lane 1: Hyperladder I

Lane 2: PGS Lo 1

Lane 3: PGS Hi 3

Lane 4: PGS Hi 4

Lane 5: Neg. Control

The only colony with an insert is PGS Hi 4. Will run a plasmid prep. However, this is the same sample that was sent for sequencing that produced nothing but vector sequence…

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Clone Restreaking – PGS2 Hi/Lo Clones (from 20110421)

Sequencing of the PGS2/COX2 clone failed (was empty vector). Restreaked bacterial clones on to a Kan50 plate (made 20110413 by SJW) from a plate that Caroline Storer had created from cloning colony selection on 20110421. Samples were labeled as PGS Lo 1 & 2 and PGS Hi 3 & 4. Additionally, there were red numbers on the plate associated with these four samples. They were 42 – 45, respectively. PGS Hi 4 (#45) was previously grown up and sequenced. This sample is what produced vector only sequence. Incubated O/N @ 37C. Hopefully we’ll bacteria is still viable and will have samples to grow up for miniprep, plamsid iso and sequencing.

Results:

Limited growth in all after O/N incubation. Will leave plate on bench over the weekend and hope to get more growth.

After the weekend on the bench, have growth in all but PGS Lo 2. Will inoculate liquid cultures for plasmid preps.

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qPCR – C.gigas GAPDH second rep on V.vulnificus exposure cDNA (from 20110311) and standard curves for COX1, COX2, GAPDH

Ran a qPCR on all cDNA samples. Created a standard curve to possibly allow for use of the BioRad software for gene expression analysis. Standard curve was created from pooled cDNA (1uL from each individual sample). Master mix calcs are here.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Standard curves aren’t that good. Will not use them. Will analyze data using PCR Miner.

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qPCR – C.gigas COX2 on V.vulnificus exposure cDNA (from 20110311)

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. Primers used were Cg_COX1/2_qPCR_F (SR ID: 1192) & Cg_COX2_454align1_R (SR ID: 1190). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Data looks good (e.g. the replicates are all very close, with the largest Cq Std. Deviation = 1.227, although this does appear to be an anomaly as the next highest Cq Std. Deviation in any of the reps is 0.633), nothing in the NTCs, & the melt curves look good. Will eventually normalize the data and then perform a complete analysis.

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qPCR – C.gigas COX1 on V.vulnificus exposure cDNA (from 20110311)

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. Primers used were Cg_COX1/2_qPCR_F (SR ID: 1192) & Cg_COX1_qPCR_R (SR ID: 1191). Samples were run in duplicate. Master mix calcs are here. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Data looks good (e.g. the replicates are all very close, with the largest Cq Std. Deviation = 0.534), nothing in the NTCs, & the melt curves look good. Will eventually normalize the data and then perform a complete analysis.

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Colony PCR – 5’RACE Colony: COX2 (repeat of yesterday’s PCR)

Repeated yesterday’s PCR on the re-streaked colony in order to run the product on a gel with a more appropriate ladder. See yesterday’s entry for all PCR info.

Results:

Lane 1: Hyperladder I

Lane 2: colony PCR

Lane 3: NTC

A band of nearly ~950bp is seen in the colony PCR, suggesting that the cloning reaction was successful. However, this does not match up with the expected size of ~1500bp seen on 20110407. Will sequence this regardless. Also, the gel on 20110407 may not have run properly (see image from that dat), which could possibly explain why we don’t see the “expected” size band of ~1500bp? Will inoculate a liquid culture for mini prep for eventual sequencing.

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Colony PCR – 5′ RACE Colony: COX2

One white colony (marked with arrow in image linked) from the two plates from Steven’s cloning (from yesterday) was picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.

Master Mix:

2x Apex Red Master Mix – 25uL

10uM M13 Forward – 1uL

10uM M13 Reverse – 1uL

H2O – 23

Added 25uL to each PCR tube.

Cycling Params:

  • 95C – 10mins

40 cycles of:

  • 95C – 30s
  • 55C – 30s
  • 72C – 2mins

1 cycle:

  • 72C – 10mins

Results:

Lane 1: Hyperladder IV

Lane 2: colony PCR

Lane 3: NTC

Turns out the Hyperladder IV (this gel was run in the Friedman Lab) only goes up to 1000bp. So, the band we see in the colony PCR reaction could be close to the expected size if the insert is present (~1500bp). Although, we also see a band in the NTC. Will repeat this PCR and run on a gel with a more appropriate ladder…

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