Plasmid Isolation – RLOv pCR2.1 Clones

Clone #1 was selected from each of the screened clones.

A sterile pipette tip was used to inoculate 5mL of 1x LBAmp100 in a 15mL conical tube. The tubes were incubated O/N @ 37C on a rocker.

3mL of liquid culture was used as input for plasmid isolation with the QIAprep Spin Mini Kit (Qiagen) according to the manufacturer’s protocol.

1mL of each culture was combined with 1mL of 50% sterile glycerol (25% glycerol final concentration) and stored @ -80C with existing bacterial stocks.

Plasmid DNA was eluted with 50μL of Buffer EB and quantified on the Roberts Lab NanoDrop1000 (ThermoFisher) in order to have a rough idea of concentrations to submit for Sanger sequencing. A dye-based quantification will be performed after sequencing results are back in order to obtain a more accurate assessment for use in ISH and/or qPCR standard curve creation.

Results:

Quality (260/280 & 260/230 ratios) look great and yields are more than sufficient. Will prep samples for Sanger sequencing.

DNase – Withering Syndrome RNA

Withering syndrome RNA, extracted from water filters, was confirmed via qPCR to contain detectable quantities of withering syndrome DNA. Before proceeding to make cDNA, residual gDNA carryover from the RNA extraction needs to be removed.

Performed DNase treatment with the Turbo DNA-free Kit (Ambion/Life Technologies), following the rigorous. Used 10μL of each of the following template RNA:

  • Day 0-1
  • Day 3-1
  • Day 7-1
  • Day 11-1

Reactions were performed in a volume of 50μL. Used 1μL of DNase for the first 30mins @ 37C and then added an additional 1μL of DNase for the final 30mins @ 37C. Samples were inactivated according to the manufacturer’s protocol and spec’d on the Roberts Lab NanoDrop1000.

Results:

Yields are consistent. The OD260/280 values are still poor (didn’t expect them to change, though). Will qPCR to verify removal of gDNA.

Plasmid Isolation – Clam RLO 16s, EHR, & EUB clones

Prepared 1x LB + 50μg/mL ampicillin. Aliquoted 5mL of LB-Amp50 liquid media to 18 15mL conical tubes. Used the restreaked plates created 20150207 and used sterile pipette tips to select each of the six positive colonies from each cloning reaction and inoculate 5mL of LB-Amp50 liquid media. Tubes were incubated O/N @ 37C on a rocker.

All cultures grew. Three milliliters from each culture were used to isolate plasmid DNA using the QIAprep Spin Mini Kit (Qiagen). Samples were eluted with 50μL Buffer EB.

Frozen bacterial stocks were made from each of the six clones, using 500μL of each bacterial culture + 500μL of sterile, 50% glycerol in 2mL screw cap tubes.

Bacterial stocks and plasmid preps were labelled in the following fashion:

  • pCR2.1/Clam RLO 16s C1 – C6
  • pCR2.1/Clam RLO EHR C1 – C6
  • pCR2.1/Clam RLO EUB C1 – C6

where “C#” indicates the clone number. The bacterial stocks were stored @ -80C in the following box “Clones Box 2″:

 

Plasmid preps were quantified on the Roberts Lab NanoDrop1000.

Results:

Spreadsheet: 20150305_ClamRLO_miniprep_ODs

After speaking with Carolyn, she decided she wanted to sequence one clone from each group. Submitted ~500ng of clone #1 (C1) from each group to GENEWIZ for Sanger sequencing (Order #: 10-290123409). Each clone was sequenced from each direction with M13F (-21) and M13R primers for a total of 6 sequencing reactions:

1       SW01    16s_01-M13F(-21)
2       SW02    16s_02-M13R
3       SW03    EHR_01-M13F(-21)
4       SW04    EHR_02-M13R
5       SW05    EUB_01-M13F(-21)
6       SW06    EUB_02-M13R

DNA Quantification & PCR – Ireland Clam S/6/14 #19 DNA

Quantification

Quantified the DNA isolated 20150130 via NanoDrop1000 (Thermo Fisher) for quick assessment of DNA.

Although the NanoDrop1000 overestimates actual yields, this is still interesting because the overall yield of this sample is greater than either of the samples isolated on 20150122, yet had significantly less starting material.

 

PCR

Ran PCRs with the following “universal” primer sets in attempt to amplify a 16s (prokaryote) fragment from the RLO that is present in this sample. Additionally, ran a universal 18s (eukaryote) primer set to verify the presence of any amplifiable DNA in the sample, in case none of the 16s primers work.

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

Master mix calcs are here: 20150203 – cPCR Clam debris DNeasy

All samples were run in duplicate.

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 2mins

Ran samples from yesterday’s PCR out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

Ladder is Hyperladder I (Bioline)

Well, ironically, the only thing that shows amplification is the no template controls (NTC) in the universal 16s primer set!  The only useful aspect of this is that it demonstrates that the reagents are functional.

The universal 18s primers don’t seem to amplify anything, either.

Tomorrow, I’ll test these primers out on DNA that I know will amplify, instead of these new clam DNA samples.

Plasmid Isolation – Withering Syndrome Phage pCR2.1/ORF20 and pCR2.1/ORF25

From cloning on 20140908, inoculated 5mL of 1x LB + 100ug/mL ampicillin with pCR2.1/ORF20 clone # 7 and pCR2.1/ORF25 clone #1. Incubated O/N @ 37C on rocker. Isolated plasmids with QIAprep Mini Kit (Qiagen) according to the manufacturer’s spin protocol, using ~3mL of culture. Eluted plasmid DNA with 100uL of EB Buffer.

Frozen bacterial stocks of each were made. 1mL of each culture was mixed with 1mL of 50% glycerol (sterile) and stored @ -80C

NOTE: Ended up diluting the pCR2.1/ORF25 by adding an additional 100uL of EB Buffer because it seemed too concentrated for proper quantification using the plate reader.

Spec’d plasmid DNA using Teacan plate reader and Pico green dye, according to lab protocol.

Results:

Spec’ing via the Teacan plate reader is not working (see separate entry regarding this).

Spec’d using NanoDrop1000:

Plasmids look great; excellent yields and quality. Will prepare some for linearization for use as standard curves and will use some for making a probe for in-situ hybridization.

Plasmid Isolation – Withering Syndrome Cultures from Yesterday

Isolated plasmid DNA from 3mL of each culture inoculated yesterday using QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer’s protocol. Eluted the plasmid DNA with 50uL of Buffer EB and spec’d on Roberts Lab NanoDrop1000.

Results:

Samples look fine; good yields, good OD260/280 ratios. Will perform NcoI restriction digests on samples to linearize them in preparation for use in a new standard curve.

RNA Isolation – Black Abalone DG Withering Syndrome Only (WSO)

Isolated RNA from 3 digestive gland (DG) samples: 08:13-7, 08:13-9, and 08:13-16 using the RNA Power Soil Kit (MoBio) according to the manufacturer’s protocol and spec’d on the Roberts Lab NanoDrop1000.

Results:

RNA is very high quality. Will prepare for submission for Illumina HiSeq sequencing. Sample pooling info is here.

RNA Isolation – Olympia Oyster Larvae from May 2012

Isolated RNA from 11 Olympia (O. lurida) oyster larvae samples, from 4 different pCO2 treatments using TriReagent (MRC) according to the manufacturer’s protocol. RNA was resuspended in 100uL of 0.1% DEPC-H2O, spec’d on the Roberts Lab NanoDrop 1000, and stored @ -80C.

Results:

RNA looks good. Will prepare for submission for Illumina HiSeq sequencing. Sample pooling info is here.

RNA Isolation – Post-Esophagus Tissue from 20120525

Isolated RNA from the fractions collected on 20120525 during the differential centrifugation procedure: Gradient Top, Gradient Junk, Gradient Bottom, Hot PE Pellet, 12:6-1 (Control PE). Samples were isolated with in 1mL TriReagent according to protocol. Samples were resuspended in 0.1% DEPC-H2O, spec’d on the Roberts’ Lab NanoDrop1000 and stored @ -80C.

Results:

The second “Gradient Junk” sample on the spec results above is actually the control 12:6-1 sample.

Overall, the sample quality (based on the OD260/280) looks poor. However, this is NOT uncommon for this tissue type (PE gland) from abalone. Yields from the control sample (12:6-1) and the Hot PE Pellet are excellent. The yields from the gradient samples are low and won’t provide enough sample for high-throughput sequencing (for which this procedure was performed). Will discuss with Steven, Carolyn and Lisa for how we want to proceed.