Tag Archives: PGS2

5’/3′ RACE – C.gigas COX2/PGS2 Nested RACE PCR

Performed nested RACE PCR on the RACE PCR products generated on 20110722 using the following nested primers: PGS2_ngspRACE_5′ (SR ID: 1350) and PGS2_ngspRACE_3′ (SR ID: 1349). Removed 2uL from each of the primary PCR reactions and brought up to 100uL in tricine EDTA (supplied in the Clontech SMARTer RACE cDNA Amplification Kit). Performed the nested RACE PCR according to the Clontech manual. Briefly, this is the same as the primary RACE PCR reaction, but using 5uL of the diluted primary PCR product and 1uL of the Nested Universal Primer (instead of 5uL of the 10X Universal Primer Mix). Master mix calcs and set up are here. Cycling params followed “Program 2″ of the Clontech protocol, modified for nested primers, and are as follows:

20 cycles:

94C 30s

68C 30s

72C 3m

Results:

Gel Layout:

Lane 1 – Hyperladder 1

Lanes 2-6 = 5′ RACE Library

Lane 2 – nGSP1 (5′ RACE primer)

Lane 3 – nGSP2 (3′ RACE primer)

Lane 4 – Neg. Control (no RACE primers)

Lane 5 – Neg. Control (nGSP1, no Universal primer)

Lane 6 – Neg. Control (nGSP2, no Universal primer)

Lane 7 – Empty

Lanes 8-12 = 3′ RACE Library

Lane 8 – nGSP1 (5′ RACE primer)

Lane 9 – nGSP2 (3′ RACE primer)

Lane 10 – Neg. Control (no RACE primers)

Lane 11 – Neg. Control (nGSP1, no Universal primer)

Lane 12 – Neg. Control (nGSP2, no Universal primer)

First of all, we see the appropriate response of each primer only producing amplicons in their respective libraries (i.e. 5′ primer only works in 5′ RACE library). This simply confirms that the primers were designed correctly. Secondly, our negative controls are clean. Thirdly, we get distinct bands from both primers. The bands marked with blue arrows in the image above were excised and purified using Ultrafree DA spin columns (Millipore). These products will be used for cloning and eventual sequencing.

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5’/3′ RACE – C.gigas COX2/PGS2 RACE PCR

Additional RACE using gene specific primers (SR IDs: 1347 & 1348) for C.gigas COX2/PGS2 according to Clontech’s SMARTer cDNA RACE Kit protocol. 3’/5′ RACE cDNA libraries are from 20080619. Master mix calcs and set up is here. Cycling params followed “Program 2″ of the Clontech protocol and are as follows:

25 cycles:

  • 94°C 30 sec
  • 68°C 30 sec
  • 72°C 3 min

Reactions were run with both primers on both libraries, just to ensure that in case there was any confusion in primer design. When finished, I will remove 2uL of the PCR reaction for use in a nested PCR reaction. Will run a gel with both sets of products, once the nested PCR is completed.

Results:

Gel Layout:

Lane 1 – Hyperladder 1

Lanes 2-6 = 5′ RACE Library

Lane 2 – GSP1 (5′ RACE primer)

Lane 3 – GSP2 (3′ RACE primer)

Lane 4 – Neg. Control (no RACE primers)

Lane 5 – Neg. Control (GSP1, no Universal primer)

Lane 6 – Neg. Control (GSP2, no Universal primer)

Lane 7 – Empty

Lanes 8-12 = 3′ RACE Library

Lane 8 – GSP1 (5′ RACE primer)

Lane 9 – GSP2 (3′ RACE primer)

Lane 10 – Neg. Control (no RACE primers)

Lane 11 – Neg. Control (GSP1, no Universal primer)

Lane 12 – Neg. Control (GSP2, no Universal primer)

As has generally been the case, our primary RACE PCRs failed to produce any products. This is why I performed the nested PCR (described above) before viewing the results of this primary PCR.

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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 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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