PCR – RLOv Clones

Colony PCRs were performed on each of the transformations from 20151015 (RLOv_ DNA_helicase, RLOv_head_to_tail, RLOv_membrane_gene_1, RLOv_membrane_gene_2, RLOv_tail_to_fiber) to confirm successful ligations in plasmid pCR2.1 using the M13F/R vector primers.

Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp100+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.

Master mix calcs are here (Google Sheet): 20151019 – Colony PCRs RLOv

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

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

Results:

 

All the PCRs look good. All white colonies selected contain a PCR product of appropriate size (i.e. larger than the blue colonies; negative [-C] control). Will select clones #1 from each to grow up for plasmid prep.

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.

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.