Tag Archives: COX2

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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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.

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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.

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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.

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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.

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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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