Tag Archives: prostaglandin synthase

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.

Share

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.

Share

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:

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.

Share

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.

Share

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.

Share

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.

Share

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.

Share

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…

Share