20111202

Bioinformatics - Identification of PGS2 through Comparison to Existing Sequence Databases

Took a number of steps to try to gather additional sequence information on the potential existence of PGS2 in C.gigas. All analyses described below were done with CLC Genomics.

1. Reference Assembly: Sigenae v8 mapped to PGS1 (FJ375303); default settings 0.8 sim. Results: One contig mapped, AM856036, which is 100% identical to FJ375303. Also, reference assemblies using lower sim values (0.75 and 0.5) do not alter the results.

2. Reference Assembly: C.gigas 454 libraries (SRR074289, SRR074290; from DATE) mapped to PGS1 (FJ375303); default settings 0.8 sim. Results: Nine total reads mapped (FU6OSJA02H48L7, FU6OSJA02HWRB7, FV2TRRU02GA34K, FU6OSJA02ICX8I, FU6OSJA02FNBOV, FV2TRRU02INIR2) that show significant differences in sequence from the reference, however none of the reads map 5' to base 837.

3. Reference Assembly: Sigenae v8 & S.gigas 454 libraries (SRR074289, SRR073290; from DATE) mapped to PGS1 (FJ375303); default settings 0.8 sim. Results: The same nine reads and the same one contig from the two previous assemblies map to the reference sequence.

4. De novo Assembly: 10 sequences discovered from the previous assemblies above; default settings. Results: 640bp contig.

5. Reference Assembly: Sigenae v8 & S.gigas 454 libraries (SRR074289, SRR073290; from DATE) mapped to 640bp contig from above; default settings 0.8 sim. Results: 24,881 reads map.


20111014

Sequencing - COX/PGS Clones from yesterday/today

Samples were submitted for sequencing to the University of Washington HTGU, two times from each direction using vector M13F/R primers. See Sequence Log for plate layout.


20111013

Mini-preps - COX/PGS Cloning Colonies from today

Selected 10 colonies (1-8, 18, 28) for mini-preps. Inoculated 5mL 1x LB + 50ug/mL of Kanamycin. Incubated O/N, 37C, 200RPM. 3mL of each culture were used for mini-preps. Used Qiagen kit. Samples were eluted w/30uL of EB.

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:

external image 20111013-01.JPG

external image 20111013-02.JPG

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.


20111012

Cloning - Purified COX/PGS "qPCR Fragment" from 20111006

Cloned the purified "qPCR Fragment" from 20111006 using the TOPO TA Cloning Kit (Invitrogen). Performed a half reaction (total volume = 3uL), using 1uL of purified PCR product. Incubated at RT for 20mins and then placed reaction on ice. Transformed chemically competent TOP 10 cells (Invitrogen) and heat shocked at 42C for 30s. Added 250uL of RT S.O.C. medium and incubated at 37C, 200RPM. Plated cells on pre-warmed Kan50+X-Gal plates (plates from 20110726; X-Gal added ~30mins before plating cells). Incubated plates O/N, 37C.


Results:

Good number of white colonies (>30). Will screen each colony with both qPCR primer sets to see if we can differentiate between the two COX/PGS isoforms in these clones.




20111006

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:

external image 20111007-01.jpg

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.


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:

external image 20111006-01.JPG

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.


20110929

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:

external image 20110930%20DNA-01.JPG

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.


20110921

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:

external image 20110922-01.JPG

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.



Ethanol Precipitation - Purified PGS1 PCR from yesterday

Added 0.1 vols of 3M sodium acetate (pH = 5.2; 38uL) and 2 vols of 100% EtOH (836uL). Incubated 30mins @ -20C. Pelleted DNA 16,000g, 15mins, 4C. Removed supe. Washed pellet w/1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Removed supe. Air-dried pellet. Resuspended in 50uL of Qiagen EB Buffer and spec'd.

Results:
Insufficient quantity of DNA for sequencing. Will re-run PCR, but will use some of this purified DNA as template to see if that helps increase yields.


20110920

PCR - Full-length PGS1 cDNA

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


Results:

external image 20110921-01.JPG

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.



20110914

PCR - Full-length PGS2 cDNA

Repeated PCR from 20110825 to attempt to amplify the full-length cDNA for PGS2 (COX2), however this time using a more robust polymerase (Amplitaq Gold) in hopes of getting results. Additionally, tried 3 different Mg2+ concentrations (1.5mM, 2.0mM, and 3.0mM). Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues. PGS2 primers = 1376, 1375.

PGS2 expected size = ~2500bp

Results:

external image 20110914-01.JPG

Loading order doesn't matter, as there are no bands. Ladder is Hyperladder I (Bioline). Will continue current sequence analysis and potentially design a new set of primers...




20110825

PCR - Full-length PGS2 cDNA

Repeated the PCR from earlier today to attempt to amplify the full-length cDNA for PGS2 (COX2). All set up was identical to what was done earlier today, with the exception of changing the annealing temp of the cycling params to 50C instead of 55C.


Results:


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:

external image 20110825-01.jpg

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).



20110811

qPCR - C.gigas V.vulnificus Exposure cDNA (from 20110311)

Ran a qPCR using 3hr Vibrio vulnificus exposure cDNA from 20110311. Original experiment conducted on 20110111 with defensin primers (SR IDs: 1109 & 1070) and GAPDH (SR IDs: 1172 & 1173). Master mix calcs are here. Cycling params, plate layout, etc can be seen in the qPCR Report (see Results). This was performed to help Herschel.

Results:
qPCR Report (PDF)
qPCR Data File (CFX96)

Initial glance at data looks good. GAPDH exhibits highly consistent Cq values across all samples, controls and exposed. Although, there is slight amplification of something in the two water samples for GAPDH, the melt curve shows that this product has a different melting temperature than our intended target. As such, I believe the GAPDH data to be useable, since no other samples exhibit this smaller product. Defensin shows clean water sample and clean melt curves with a single peak. However, it seems like we may not see an effect on defensin expression in response to the Vibrio vulnificus exposure...


20110804

Sequencing - C.gigas COX2/PGS2 Clone #4 from 20110728

Used new primers for sequencing (SR IDs: 1351 & 1352) clone #4. Sequenced clone two times in each direction. DNA and primers were sent for sequencing at ASU. Requested "High GC" treatment to help overcome the issue seen on 20110728.

Results:
Sequencing results received 20110810. Initial analysis suggests that we managed to fully sequence this clone! Will try to assemble a full-length CDS for COX2/PGS2.


20110728

Plasmid Isolation & Sequencing - C.gigas COX2/PGS2 Clones (from yesterday)

Isolated plasmid DNA from 3mL of liquid cultures that were inoculated yesterday using Qiagen's miniprep kit. DNA was eluted with 50uL of EB. DNA was prepped and sent for sequencing to ASU sequencing facility. Each clone was sequenced two times in each direction. Samples are as follows:

Name - Clone # Primer
SJW01 - 1 M13F
SJW02 - 1 M13F
SJW03 - 1 M13R
SJW04 - 1 M13R
SJW05 - 2 M13F
SJW06 - 2 M13F
SJW07 - 2 M13R
SJW08 - 2 M13R
SJW09 - 3 M13F
SJW10 - 3 M13F
SJW11 - 3 M13R
SJW12 - 3 M13R
SJW13 - 4 M13F
SJW14 - 4 M13F
SJW15 - 4 M13R
SJW16 - 4 M13R

Clone #s are as follows:
1 - 5' Library Top band
2 - 5' Library Mid band
3 - 5' Library Bottom band
4 - 3' Library band


Results:
Sequencing results received 20110801. SJW15 and 16 apparently stop abruptly. The sequencing facility believes this to be caused by secondary structure of the template. Depending on how things align, I may consider using 7-daeza-GTP in a PCR reaction and re-sequencing this clone, as the 7-daeza-GTP helps relax secondary structure.

Spoke with Steven and he suggested just designing new primers closer to each other and resubmit.



20110727

Bacterial Cultures - C.gigas COX2/PGS2 Clones (from yesterday)

Inoculated 4 x 5mL 1xLB + Kan50 with a colony from each set of clones, incubated 37C, 200RPM, O/N. Will mini prep and send for sequencing tomorrow.


20110726

Colony PCRs - C.gigas COX2/PGS2 Clones (from yesterday)

Performed colony PCRs on the 4 sets of cloning reactions that were performed yesterday using the M13F/R vector primers. Colonies were picked, restreaked on a fresh LB Kan50 plates (made 20110726 by SJW) and PCR'd. Master mix calcs are here. Selected 8 white colonies from each cloning reaction for PCR. Restreaked plate was incubated @ 37C O/N.


Cycling Params:

95C - 10m
40cycles of:
95C - 10s
55C - 10s
72C - 3m

Results:

external image 20110727-01%20Gel.jpg

external image 20110727-02%20Gel.jpg
Hyperladder I is used as the ladder in both gels.

Cloning results look great (except Colony #1 in the 5' Top Band didn't produce a product). Will select a re-streaked colony from each set and inoculate liquid culture for mini prep and subsequent sequencing.




20110725

Cloning - C.gigas COX2/PGS2 5'/3' RACE Products (from earlier today)

The bands that were excised and purified earlier today were cloned in to pCR2.1 using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer's protocol with the following changes: used 4uL of all PCR products, incubated ligation reactions for 15mins @ RT, incubated competent cells with ligation reactions for 15mins on ice.

Two volumes of each reaction were plated (50uL and 100uL) on Kan50 plates with X-gal (made 20010412 by SJW) and incubated @ 37C O/N.

Results:

Ample number of white colonies for all 4 sets of cloning reactions.


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:

external image 20110725-02%20Gel%20marked.jpg

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.




20110722

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:

external image 20110725-01%20Gel.jpg

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.




20110715

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:

external image 20110721%20PGS2%20BLAST%20Alignment.png

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.



20110712

NanoDrop1000 Calibration Check

Performed the calibration check procedure according to the manufacturer's protocol. The ND1000 calibration procedure indicated that the accuracy of the ND1000 has drifted more than the acceptable 5% and requires calibration by a professional. Will get a price quote and speak with Steven.

Plasmid Isolation - Miniprep on PGS Hi 4 Colony from yesterday

Plasmid was isolated from 3mL of liquid culture started yesterday using the Qiagen MiniPrep Kit, according to the manufacturer's protocol. Will send off for sequencing.




20110711

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:
external image 20110712-01.jpg

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...




Bacterial Cultures - Liquid Cultures of PGS2/COX2 Colonies from 20110707

Inoculated 5mL of 1xLB + Kan50 with re-streaked colonies from 20110707. Incubated O/N, 37C, 200RPM. Will isolated plasmids of those with inserts tomorrow.


20110707

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.