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 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.

 

Ligation – EcoRI-digested WS Phage and EcoRI-digested pCR2.1 (from 20121204)

Performed a traditional ligation using the EcoRI-digested WS phage and pCR2.1. An aliquot of the digested pCR2.1 was diluted 1:10 in an attempt to reduce self-ligation due to the significant difference in quantity of DNA between the phage and vector.

Reaction:

  • EcoRI WS Phage – 5uL
  • EcoRI pCR2.1 (diluted 1:10) – 1uL
  • 10x Ligase Buffer (NEB) – 1uL
  • T4 DNA Ligase (NEB) – 1uL
  • H2O – 2uL

Incubated reaction at 16C for 24hrs to maximize ligation. Sample was stored at -20C.