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.

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.

Cloning – Purified Clam RLO PCRs

Purified PCR products from 16s, EUB, and EHR primers were used for cloning.

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

LIGATION

Ligation reactions:

  • PCR product: 4μL
  • 10x Buffer: 1μL
  • Vector (pCR2.1): 2μL
  • Water: 1μL
  • T4 Ligase: 1μL

Incubate O/N (~18hrs) @ 14C.

LB-Amp50 plates from yesterday were used.

TRANSFORMATION

40μL of X-gal (40mg/mL) was added to a LB-Amp50 plate, spread and warmed @ 37C.

Three 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-Amp50+X-gal plates, spread and incubated O/N at 37C.

 

Cloning – Purified Clam RLO PCR

Purified PCR product (universal ehrlichia primers) from 20150219 was used for cloning.

Purified PCR volume: 57μL

Purified PCR amount: 75ng (estimated from ladder on gel)

Purified PCR conc: 1.3ng/μL (calculated from numbers above)

The PCR product was ligated using the TA Cloning Kit (Invitrogen).

LIGATION

Ligation reaction:

  • PCR product: 4μL
  • 10x Buffer: 1μL
  • Vector (pCR2.1): 2μL
  • Water: 1μL
  • T4 Ligase: 1μL

Incubate O/N (~18hrs) @ 14C.

Lysogeny broth (LB) plates were made: (100mL of 1x LB) containing 1.5% agar (1.5g), autoclaved, cooled, 500μL of 20mg/mL ampicillin added (50μg/mL final concentration), mixed, and poured.

TRANSFORMATION

40μL of X-gal (40mg/mL) was added to a LB-Amp50 plate, spread and warmed @ 37C.

A vial 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. The vial was heat shocked @ 42C for 30s and immediately transferred to ice. The vial of cells was transferred to the LB-Amp50+X-gal plate, spread and incubated O/N at 37C.

 

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.