Tag Archives: PCR

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.

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.

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.

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:

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…

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).

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.

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…

Colony PCR – Colonies from COX1 Genomic Cloning (from 20110411)

Ran colony PCR on various colonies produced from cloning on 20110411. All colonies were picked, re-streaked on Kan50 plate(s) and PCR’d. Master mix calcs are here. Cycling params:

  • 95C – 10mins

40cycles of:

  • 95C – 30s
  • 55C – 30s
  • 72C – 3mins
  • 72C – 10mins

Results:

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.

Colony PCR – 5′ RACE Colony: COX2

One white colony (marked with arrow in image linked) from the two plates from Steven’s cloning (from yesterday) was picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.

Master Mix:

2x Apex Red Master Mix – 25uL

10uM M13 Forward – 1uL

10uM M13 Reverse – 1uL

H2O – 23

Added 25uL to each PCR tube.

Cycling Params:

  • 95C – 10mins

40 cycles of:

  • 95C – 30s
  • 55C – 30s
  • 72C – 2mins

1 cycle:

  • 72C – 10mins

Results:

Lane 1: Hyperladder IV

Lane 2: colony PCR

Lane 3: NTC

Turns out the Hyperladder IV (this gel was run in the Friedman Lab) only goes up to 1000bp. So, the band we see in the colony PCR reaction could be close to the expected size if the insert is present (~1500bp). Although, we also see a band in the NTC. Will repeat this PCR and run on a gel with a more appropriate ladder…