PCR – RLOv for Cloning & Sequencing

After yesterday’s confirmation that the qPCR primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv, I needed to generate PCR products to clone and sequence.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

All samples were run in duplicate.

Master mix calcs are here: 20151009 – PCR RLOv

Cycling Params (PTC-200; MJ Research)

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 95
  • 600
40 Cycles
  • 95
  • 55
  • 72
  • 15
  • 15
  • 30

Samples were run on a 0.8% agarose 1x TBE gel, stained with ethidium bromide.

Results:

Amplification looks great. No amplification in no template controls (NTCs). Excised bands and purified products using Ultrafree DA Spin Columns (Millipore). Samples will be stored @ 4C until I am able to clone them for sequencing.

 

Gel image showing excised bands. And, it’s a complete hack job, which is embarrassing…

Agarose Gel – Phage ISH Primers PCRs

Ran PCR products from yesterday on a 1% agarose 1x TBE gel, stained with ethidium bromide.

Results:

IMPORTANT NOTE: The negative control sample should actually be labelled UW08:22-11A.

 

PRIMER SET EXPECTED PCR SIZE (bp) RESULT SIZE (bp)
RLOv_membrane_gene_1 401 ~400bp
RLOv_membrane_gene_2 318 ~400bp
RLOv_tail_fiber_gene 451 ~500bp

PCR looks great. Excellent amplification in the RLO positive samples (06:6-54), with no amplification in the negative controls (UW08:22-11A) nor in the no template controls (NTC).

Excised the bands from each of the RLOv positive samples (see gel image below) and purified the DNA using UltrafreeDA Spin Columns (Millipore) according to the manufacturer’s protocol. DNA was stored @ 4C for cloning/labelling/sequencing at a later date.

Gel image showing excised regions.

PCR – Ireland Clam RLO DNA S/6/14 #19 (from 20150130)

After previously confirming that the issue with previous PCRs was due to bad reagents, I re-ran the PCR on the clam RLO DNA isolated 20150130 using a set of universal 16s primers, as well as a universal 18s primer set to serve as a positive control that amplifiable DNA was present in the sample.

Master mix calcs are here: 20150219 – cPCR Universal Primers Apex Red MM

Primers being used are:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 1mins

Samples were run on 1.0% agarose, low TAE gel stained w/EtBr.

Results:

Ladder used was O’GenRuler 100bp DNA Ladder (Thermo-Fisher).

No sample was loaded directly next to ladder to facilitate excision, if necessary.

Each sample was accompanied by a no template control (NTC).

The ehrlichia universal primers (EHR) and the universal 18s (18s) primers are the only two primer sets that do not have contamination present in the NTCs.

Excised the EHR band and purified with Ultrafree-DA columns (Millipore). Purified DNA was stored @ -20C and will be used for cloning/sequencing next week.

Have already ordered additional primer sets of those above that are contaminated. Will re-run the PCR with those new, sterile primer sets when they arrive to obtain a larger product (the EHR amplicon is only ~350bp).

PCR – Withering Syndrome Phage

We received MiSeq data back from Stan Langevin (samples submitted 20140717) and he believes he has sequenced the entire WS phage. Carolyn and Colleen designed some primers on two of the open reading frames annotated by Stan. Ran PCR with the three primer sets to test out:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Master mix calcs are here: 201400813 – PCR WS phage

Cycling params:

Ran samples on 1.2% 1x TBE + EtBr.

Results:

Ladder: O’GeneRuler 100bp DNA Ladder (ThermoFisher)

Good amplification from all three primer sets. The pinto abalone sample (UW08:22-65) that should be naive for withering syndrome and phage did not amplify as expected.

Excised bands from each primer set in the 06:6-41 group and purified using Ultrafree DA spin columns (Millipore). Will save for potential cloning usage, depending on future results.

Chloroform Cleanup – EcoRI-digested Withering Syndrome Phage DNA from earlier today

To concentrate the sample, a chloroform cleanup and EtOH precipitation was performed. An equal volume of chloroform was added to the sample (220uL), vortexed for 30s and spun @ 16,000g at 4C for 15mins. The aqueous phase was transferred to a clean tube for EtOH precipitation.

0.1 volumes (16.8uL) of 3M NaOAC (sodium acetate; pH = 5.2) and 2.5 volumes (462uL) of ice cold 100% EtOH were added to the sample and mixed thoroughly. The sample was incubated O/N at -20C. The following day the sample was pelleted by spinning at 16,000g at 4C for 30mins. As expected, there was no visible pellet due to the extremely low quantity of DNA in the sample. The supernatant was discarded, the pellet was gently washed (no mixing/vortexing) with 70% EtOH, and spun @ 16,000g at 4C for 15mins. The supernatant was discarded the pellet was air dried for 15mins. The sample was reconstituted in 10uL of Buffer EB (Qiagen), which is simply 10mM Tris-HCl, and stored at 4C.

Gel Purification – EcoRI-digested pCR2.1 Vector from earlier today

Gel-excised band from earlier today was purified using the Ultra DA-free (Millipore) spin column according to the manufacturer’s protocol. Purified DNA was stored at 4C.

Restriction Digestions – Withering Syndrome Phage DNA and p16RK3

Performed restriction digestions on Phage RLO DNA (isolated on 20121130) and p16RK3 using EcoRI from NEB.

Phage RLO Digest

  • DNA – 192uL
  • 10x EcoRI Buffer – 22uL
  • EcoRI – 4uL
  • H2O – 2uL
  • TOTAL = 220uL

p16RK3 Digest

  • DNA (1ug) – 5.39uL
  • 10x EcoRI Buffer – 5uL
  • EcoRI – 1uL
  • H2O – 38.61uL
  • TOTAL = 50uL

Samples were incubated at 37C for 1hr. The p16RK3 sample was run on a 0.8% TBE gel to confirm digestion and isolate the vector-only band from the sample. The Phage RLO sample was subject to a chloroform cleanup and EtOH precipitation.

Results:

Digestion of Phage RLO DNA was not verified due to the extremely low quantities of DNA. In order to minimize loss of material, we will trust that the digestion was successful if the digestion of p6RK3 was successful.

Gel Loading

Lane 1 – Hyperladder I (Bioline)

Lane 2 – p16RK3 EcoRI

Neglected to run undigested p16RK3. However, the digestion pattern looks correct. The empty vector (pCR2.1; Invitrogen) should be 3931bp and we see the largest molecular weight band of the digest is running at ~4000bp. That band was excised and will be purified for subsequent ligation.

PCR – Cloning AF133090 for New RLP Standard Curve

Ran PCR using newly ordered primers (WS_16s_1_F, WS_16s_1501_R) that should amplify the entirety of AF133090 in order to create a new RLP plasmid standard curve for the RLP qPCR assay. Master mix calcs are here. Template used was “Big Reds” fecal DNA extraction by Lisa on 4/3/2012. Sample was run on a 0.8% TBE gel.

Results:

Lane Loading Guide:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – AF133090 PCR

Lane 3 – NTC

Success! The bright band in the Af133090 sample is at exactly 1500bp, as expected. This band was excised and purified using an Ultra-DA column (Millipore). The extensive smearing in this lane is most likely due to the template used, which consists of a DNA extract from abalone feces. This sample likely contains a high amount of DNA, which results in the high molecular weight smearing seen.