Tag Archives: pCR2.1/RLOv_tail_fiber_gene

PCR – RLOv In-situ Hybridization (ISH) Probes

Ran probe-labeling PCRs to use in in-situ hybridization (ISH) using the PCR DIG Probe Sysnthesis Kit (Roche). Generated PCR probes for using the following BamHI-linearized plasmids:

  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_tail_fiber

The Roche protocol recommends using only 10pg of plasmid DNA for probe labelling. As such, all three probes were diluted 1:10,000. A 1:1000 (999μL H2O + 1μL of plasmid) was made first. Then a 1:10 dilution was made (90μL H2O + 10μL from 1:1000 dilution of plasmid).

Additionally, I ran half reactions to conserve kit components. Roche recommends 50μL reactions; I ran 25μL and scaled all components appropriately.

All reactions were set up on ice and run in 0.2mL strip-cap PCR tubes.

Reaction calculations are here (Google Sheet): 20151109 – RLOv ISH Probe PCRs

Cycling params:

  1. 95C – 5mins
  2. 95C – 15s
  3. 55C – 15s
  4. 72C – 30s
  5. Go to Step 2, repeat 39 times.
  6. 72C – 10mins

After the PCR, 5μL of each reaction was run on a gel.


Hyperladder I (Bioline)

PCR DIG probe labelling products run on 1.1% agarose 1x TBE gel stained w/EtBr. A ‘+’ indicates DIG reaction, while a ‘-‘ indicates no DIG in reaction.

Two reactions were run for each plasmid: one with the DIG label (indicated by a ‘+’) and one without (indicated by a ‘-‘). If the labeling was successful, the PCR products from those reactions containing DIG will be larger (i.e. migrate slower) than those without. That is exactly what we see in each of the three potential ISH targets.

So, we now have three ISH probes ready for action! Will proceed with making fresh ISH buffers and ISH.

Probes were transferred to 0.5mL snap cap tubes and stored in my -20C box.


DNA Quantification – BamHI-Linearized pCR2.1/RLOv plasmids

Quantified the linearized RLOv plasmids using the Quant-It DNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards (10μL each of 0, 5, 10, 20, & 40ng) were run in triplicate.

Linearized plasmids were quantified in replicates of six.

Quantification was performed in black 96-well plates in the Seeb Lab Victor3 1420 (Perkin Elmer) plate reader. See the spreadsheet linked in the Results below for reading protocol.


Calculations & raw fluorescence readings (Google Sheet): 20151106_quantification_RLOv_linearized_plasmids

Standard Cuve R^2 = 0.999

Best Fit Equation: y = 1174.8215x + 8919.308333333

pCR2.1/RLOv_DNA_helicase 15.498
pCR2.1/RLOv_head_to_tail 17.887
pCR2.1/RLOv_membrane_gene_1 25.111
pCR2.1/RLOv_membrane_gene_2 28.264
pCR2.1/RLOv_tail_fiber 23.442

Will proceed to making qPCR standard curves from the DNA helicase and the head-to-tail linearized plasmids.


Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20


Digestion Master Mix

Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.


Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).



Sanger Sequencing Submission – pCR2.1/RLOv Clones

Submitted the pCR2.1/RLOv clones from earlier this week for Sanger sequencing to Genewiz (order #10-313205054).

Submitted ~500ng of each plasmid in a final volume of 15μL (including primer). Each clone will be sequenced from each direction with M13F (-21) (25pmol; 2.5μL of 10μM stock) and M13R primers (25pmol; 2.5μL of 10μM stock) for a total of 10 sequencing reactions:

  • SW01    RLOv_DNA_Helicase-M13F_-21_
  • SW02    RLOv_head_to_tail-M13F_-21_
  • SW03    RLOv_membrane_gene_1-M13F_-21_
  • SW04    RLOv_membrane_gene_2-M13F_-21_
  • SW05    RLOv_tail_fiber-M13F_-21_
  • SW06    RLOv_DNA_Helicase-M13R
  • SW07    RLOv_head_to_tail-M13R
  • SW08    RLOv_membrane_gene_1-M13R
  • SW09    RLOv_membrane_gene_2-M13R
  • SW10    RLOv_tail_fiber-M13R

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.


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