Tag Archives: PGS1

PCR – COX/PGS Cloning Colony Screens from yesterday

Performed PCR on 40 colonies using both qPCR primer sets to see if I could differentiate between which colonies potentially contained each isoform to reduce the amount of clones needed for sequencing. Master mix and cycling params are here. Primers used were:

Cg_COX1/2_qPCR_F (SR ID: 1192)

Cg_COX1_qPCR_R (SR ID: 1191)

Cg_COX2_454align1_R (SR ID: 1190)

Positive controls for both primers set were also run. The positive control template was the purified PCR product from 20111006.

Results:

Ladder is Hyperladder II (Bioline). Samples are loaded, left to right, as PGS1 and PGS2 on each colony (e.g. on the bottom gel image, under the “Colony 40″ label is the PGS1 rxn on the left and the PGS2 rxn on the right).

Nearly every colony exhibits amplification using both primer sets, w/the PGS1 reaction producing a band of ~250bp and the PGS2 reaction producing a band of ~750bp. Colonies 18 and 28 are an exception to this and produced no band with the PGS2 primer set. NTCs were clean. The positive controls worked as expected, yielding a band of ~250bp for PGS1 and a band of ~250bp for PGS2.

It is confusing as to why the size of the PGS2 positive control is different than the product that was generated from the colony PCRs.

Will select 10 colonies for mini-preps.

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PCR – Purified COX/PGS 1/2 DNA from earlier today

Ran PCR using primers Cg_COX1/2_qPCR_F, Cg_COX1_qPCR_R, Cg_COX2_454align1_R (SR IDs: 1192, 1191, 1190; respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These reactions will verify (sort of) if we have both isoforms present in the PCR performed earlier today, prior to cloning. Master mix calcs and cycling params are here.

Results:

Lane 1: Hyperladder I (Bioline)

Lane 2: COX1/PGS1 primer set

Lane 3: COX1/PGS1 primer set NTC

Lane 4: COX2/PGS2 primer set

Lane 5: COX2/PGS2 primer set NTC

NTCs are clean for both primer sets. We see bands of the expected size for both primer sets. Additionally, we see lower expression in COX2/PGS2, as we observed in our previous qPCR reactions with these primer sets. Will clone the large fragment that was PCR’ed/purified from earlier today.

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PCR – Region Outside of COX/PGS qPCR Primers

Ran PCR using primers Cg_COX_982_F and Cg_COX_2138_R (SR IDs: 1149 & 1151, respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These primers anneal 5′ and 3′ of where the qPCR primers for both COX1/PGS1 and COX2/PGS2 anneal. Master mix calcs and cycling params are here. Ran multiples of the same reaction to ensure sufficient product for use in cloning/PCR.

Results:

Gel is loaded with Hyperladder I (Bioline) and 7 samples (no NTC; don’t ask). Band in each lane is of the expected size (~1200bp). Each band was excised and purified using Ultra-free DA columns (Millipore), according to protocol. Purified DNA will be used in a subsequent PCR using the qPCR primers for COX/PGS 1&2 BEFORE cloning this product for sequencing.

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Ethanol Precipitation – Full-length PGS1 cDNA (from 20110921)

Performed an EtOH on gel-purified PCR products from 20110921. Briefly, added 0.1 vols of 3M sodium acetate (pH=5.2; 43uL), mixed and then added 2.5 vols of 100% EtOH (1182.5uL). Mixed, split into two tubes (due to high volume not fitting in a single tube) and incubated @ -80C O/N. Pelleted DNA 16,000g, 20mins, 4C. Discarded supe. Washed pellet w/ 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended both pellets in a TOTAL of 25uL Qiagen Buffer EB (10mM Tris-HCl) and spec’d.

Results:

Now have sufficient DNA for sequencing.

What’s next?

Generate PCR product using primers that anneal OUTSIDE of each of the qPCR primers and then sequence those bands to ensure that the qPCR primers are actually annealing to two different isoforms.

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PCR – Full-length PGS1 cDNA

Still have insufficient quantities of DNA for sequencing. Master mix calcs and cycling params are here. Additionally, used some of the purified PCR product as template in one of the reactions, just for comparison purposes. cDNA template was pooled cDNA from 20110311 from various C.gigas tissues. Also, increased the amount of template 4-fold in an attempt to obtain higher yields of PCR products for sequencing.

Results:

Lane 1: Hyperladder I (Bioline)

Lane 2: PCR 1 (cDNA template)

Lane 3: PCR 2 (cDNA template)

Lane 4: PCR 3 (PCR template)

Lane 5: Neg. Control

Bands were excised and will be purified using Ultra-free DA columns (Millipore). Also, it’s very clear that using the purified PCR product as template produced a much greater yield, although there appear to be some spurious, high-molecular weight banding/smearing.

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PCR – Full-length PGS1 cDNA

Need more PCR product for sequencing. Repeated reaction from 20110825.

Results:

Lane 1 – Hypperladder I (Bioline)

Lane 2 – PCR 1 & 2

Lane 3 – PCR 3 & 4

Lane 4 – PCR 5 & 6

Lane 5 – Neg. Control

Bands from lanes 2 – 4 were excised and purified with Ultra-free DA columns (Millipore) and spec’d. Concentration was extremely low (3.5ng/uL) and too dilute for sequencing. Will EtOH precipitate.

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PCR – Full-length PGS1 & PGS2 cDNAs

Ran PCR to amplify full-length cDNAs of PGS1 & PGS2 (COX1 & COX2) using primers designed to anneal in the 5’/3’UTRs of each isoform. PGS1 primers = SRIDs: 1377, 1378. PGS2 primers = 1376, 1375. Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues.

PGS1 Expected Size = ~2300bp

PGS2 Expected Size = ~2500bp

Results:

Gel

Lane 1 – Hyperladder I (Bioline)

Lane 2 – PGS1

Lane 3 – PGS1 NTC

Lane 4 – PGS1 NTC

Lane 5 – PGS2

Lane 6 – PGS2 NTC

Lane 7 – PGS2 NTC

PGS1 Results: PGS1 PCR produces a single band of the expected size (~2300bp), indicating that the two primers, which were designed to anneal in the 5’/3’UTRs of the gene and should be highly specific to just this isoform, work perfectly. The band was excised and stored @ -20C in “Sam’s Miscellaneous” box.

PGS2 Results: PGS2 PCR didn’t produce any product. Will repeat with a lower annealing temp (50C instead of 55C).

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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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Bacterial Cultures – Colonies Selected from Yesterday’s Colony PCRs

Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N at 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected:

  • MM09 – #1, 2, 8
  • MM11 660bp – #1, 2, 8
  • MM10 2/8/11 – #1, 2
  • MM04 1/19/11 – #2, 3
  • MM11 3000bp – #3
  • MM04 1500bp – #4
  • MM06 1/19/11 – #1, 2
  • MM04 550bp – #1, 2
  • MM05 1/19/11 – #1, 2
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