Cloning – Purified OsHV-1 ORF117 PCRs

Purified OsHV-1 ORF117 PCRs from earlier today were separately ligated using the Original TA Cloning Kit (Invitrogen).

LIGATION

Ligation reactions:

  • PCR product: 5μL
  • 5x Buffer: 2μL
  • Vector (pCR2.1): 2μL
  • T4 Ligase: 1μL

Incubate 1hr @ RT.

TRANSFORMATION

50μL of X-gal (40mg/mL) was added to a LB-Amp100 plate, spread and warmed @ 37C.

Three vials of OneShot TOP 10 chemically competent cells (Invitrogen) were thawed on ice. 5μL of the ligation reaction was added to the cells, gently mixed and incubated on ice for 5mins. Thecells were transferred to the LB-Amp100+X-gal plates, spread and incubated O/N at 37C.

Results:

All three transformations failed. All of them produced only blue colonies and very few total colonies.

The low number of colonies prompted me to look at the troubleshooting in the manual for The Original TA Cloning Kit (Invitrogen). It turns out that after six months of storage, the vector begins to lose the T overhangs. The kit I used is from 2014; three years beyond the tentative expiration date. This is likely the cause of the failed transformations.

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.

Cloning – Purified Abalone RLOv PCR Products

Purified PCR products from 20151008 (in situ hybridization [ISH] primers) & 20151009 (qPCR primers) RLOv primers were used for cloning. The qPCR primers are intended to develop a qPCR standard curve and the ISH primers are intended to develop three ISH probes.

  • RLOv_DNA_helicase (for qPCR)
  • RLOv_head_to_tail_gene (for qPCR)
  • RLOv_membrane_gene_1 (for ISH)
  • RLOv_membrane_gene_2 (for ISH)
  • RLOv_tail_fiber_gene (for ISH)

The PCR products were separately ligated using The Original TA Cloning Kit (Invitrogen).

LIGATIONS

REAGENT SINGLE REACTION VOL (μL) x 5.5
Purified PCR 5 NA
10x Ligase Buffer 1 5.5
pCR2.1 Vector 2 5.5
H2O 1 5.5
T4 DNA Ligase 1 5.5
TOTAL 10 22

Ligation reactions were set up on ice.Combined 5μL of purified PCR product with 5μL of ligation master mix in a 1.5mL snap cap tube. Incubated 24hrs @ RT.

 

TRANSFORMATIONS

50μL of X-gal (20mg/mL) was added to a LB-Amp100 plates, spread and warmed @ 37C.

Five vials of OneShot TOP 10 chemically competent cells (Invitrogen) were thawed on ice. 3μL of the ligation reaction was added to the cells, gently mixed and incubated on ice for 5mins. 50μL of cells were transferred to the LB-Amp100+X-gal plates, spread and incubated O/N at 37C.

Colony PCRs – Clam RLO 16s, EHR, EUB

Colony PCRs were performed on each of the three transformations from yesterday (16s, EHR, and EUB primers) using the M13F/R vector primers. Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp50+x-gal plate and then used to inoculate the respective PCR reactions. Six white colonies (positive clones) and a single blue colony (negative clone) were selected from each transformation.

Restreaked plates were incubated @ 37C O/N and then stored @ 4C (Parafilmed).

Master mix calcs are here: 20150227 – Colony PCR Clam RLO

30μL of each reaction was run on a 1% agarose 1x Low TAE gel, stained w/EtBr.

Results:

Ladder: Hyperladder I (Bioline)

Upper Left: 16s colonies 1 – 7

Upper Right: EHR colonies 1 – 6

Lower Left: EUB colonies 1 – 7

Based on the PCRs used for cloning, all white colonies screened exhibit the expected product sizes. Additionally, each of the blue (negative) colonies, produced the expected band size that are indicative of an empty plasmid.

Will select a positive colony from each set for mini prep and Sanger sequencing.

PCR – Withering Syndrome Bacteriophage Clone Screen

10 colonies from both ligations (pCR2.1/ORF20 & pCR2.1/ORF25) were picked with clean pipette tips, streaked on a different LB-Amp50+X-gal plate that had been gridded and numbered, and then used to inoculate the PCR reactions.

PCRs were performed with respective phage primers.

Master mix calcs are here: 20140909 – Phage Colony Screens

Cycling params:

1 cycle:

  • 95C – 10mins

40 cycles:

  • 95C – 15s
  • 55C – 15s
  • 72C – 30s

Results:

Top half of gel are the pCR2.1/ORF20 colonies.

Bottom half of gel are the pCR2.1/ORF25 colonies.

All amplified. Will select one of each from the gridded plates for plasmid isolation for use in qPCR standard curves and in-situ hybridization (ISH).

Cloning – Withering Syndrome Bacteriophage ORFs 20 and 35

Cloned purified PCR products from 20140813 into the pCR2.1 vector using The Original TA Cloning Kit (Life Technologies/Invitrogen), according to the manufacturer’s protocol. Used 2uL of PCR product. Incubated ligation at RT for 15mins. Transformed TOP10 chemically competent cells (Life Technologies/Invitrogen) following the Rapid Transformation protocol (added 4uL of ligation reaction to thawed cells; incubated on ice 5mins; plated 50uL of cells on pre-warmed LB-Amp (50ug/mL) + X-gal (40uL of 40mg/mL stock) plates). Incubated O/N at 37C.

Results:

Both cloning reactions look great; lots of potential clones. Will screen nine from each via PCR for detection of our desired insert.