Transformation – pCR2.1/OsHV-1_ORF117 into One Shot Top10 Chemically Competent Cells

Yesterday’s transformation with freshly prepared ampicillin didn’t produce any transformants, suggesting the DNA concentration is too low.

Previously, I tried to elute the DNA from one of the spots Tim sent with 50uL. This volume was enough to soak the Whatman paper and produce excess liquid. In retrospect, I think the volume was too large and diluted the DNA too much (concentration wasn’t measurable via Qubit)

Today, I eluted with 25uL. Since this volume was too little to produce excess liquid, I created a spin “filter” to extract the absorbed liquid. Briefly, I punctured the top and bottom of a 0.5mL snap cap tube with an 18 gauge needle, inserted the Whatman paper disc into this tube, and then put this tube in a 2mL snap cap tube. This assembly was spun @ 18,000g RT for 3 mins.

Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol (thaw cells on ice, add DNA, incubate 5mins, plate on pre-warmed ampicillin plates).

Cells were plated on pre-warmed (37C) LB Amp100 plates.

Plates were incubated overnight at 37C.

Results:

Wow, only two colonies! Well, as they say, you only need one. Will PCR, re-streak, and inoculate 5mL liquid cultures to see if either of these colonies seem to have the insert.

Transformation – pCR2.1/OsHV-1_ORF117 into One Shot Top10 Chemically Competent Cells

This is a repeat since the previous attempt at obtaining sufficient quantities of plasmid for sequencing failed. Although I’m not sure why, I figure it’s easy enough to re-do using ampicillin stocks that aren’t many years old. :)

The old ampicillin may not have been strong enough to put enough selective pressure on transformants, which possibly led to such little plasmid recovery.

I prepared fresh ampicillin solution (20mg/mL) and made new LB plates (ampicillin concentration 100ug/mL).

Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol (thaw cells on ice, add DNA, incubate 5mins, plate on pre-warmed ampicillin plates).

Cells were plated on pre-warmed (37C) LB Amp100 plates.

Plates were incubated overnight at 37C.

Results:

No transformants. So, this suggests that the original ampicillin was bad. Now, the lack of transformants suggests the plasmid concentration is too low. Will try eluting the DNA from the second spot of Whatman paper.

Transformation – pCR2.1/OsHV-1_ORF117 into One Shot Top10 Chemically Competent Cells

Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol.

Cells were plated on pre-warmed (37C) LB Amp100 plates with X-gal.

Plates were incubated overnight at 37C.

Results:

Looks good – ample colonies and no blue colonies (blue colonies = empty vector). Will screen a subset of the colonies via cPCR.

 

 

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.