Tag Archives: bacterial transformation

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.

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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.

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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.

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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.

 

 

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