Tag Archives: pCR2.1

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.

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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.

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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.

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Cloning – C.gigas COX2/PGS2 5’/3′ RACE Products (from earlier today)

The bands that were excised and purified earlier today were cloned in to pCR2.1 using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s protocol with the following changes: used 4uL of all PCR products, incubated ligation reactions for 15mins @ RT, incubated competent cells with ligation reactions for 15mins on ice.

Two volumes of each reaction were plated (50uL and 100uL) on Kan50 plates with X-gal (made 20010412 by SJW) and incubated @ 37C O/N.

Results:

Ample number of white colonies for all 4 sets of cloning reactions.

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Ligations – COX1/COX2 PCR Products

Performed ligations/cloning on a variety of COX1 genomic and COX 5′ RACE products using the TOPO TA Cloning Kit (Invitrogen). Used 2uL of gel-purified PCR product in each cloning rxn, 2.5uL of H2O, 1uL of salt solution, and 0.5uL of the Invitrogen pCR2.1 vector. Incubated samples for 5mins at RT. Used 2uL of the cloning reaction to transform TOP10 chemically competent cells (Invitrogen), mixed very gently, incubated on ice for 5mins, heat shocked at 42C for 30s and immediately placed on ice. Added 250uL of SOC Medium and incubated tubes at 37C, 200RPM for 1hr. Plated 50uL of each transformation on LB+Kan plates (made by Steven on unknown date with unknown Kan concentration) containing 40uL of 40mg/mL X-gal. Incubated O/N at 37C. Remaining volume of transformed bacteria were stored @ 4C.

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