Received the sample described above (and pictured below) from Tim Green via Colleen Burge. Plasmid was sent dried, on filter paper. Will elute plasmid from one circle of filter paper with 50uL of TE buffer.
Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).
|PLASMID||Vol for 1.5μg (μL)||H2O to 40μL|
Digestion Master Mix
|REAGENT||SINGLE REACTION (μL)||x 5.5 (μL)|
|10x Buffer 3.1 (NEB)||5||27.5|
|TOTAL||50||Add 10μL to each tube|
Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.
Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.
Besides the funky way this gel ran, the digests look to be complete.
Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).
Submitted the pCR2.1/RLOv clones from earlier this week for Sanger sequencing to Genewiz (order #10-313205054).
Submitted ~500ng of each plasmid in a final volume of 15μL (including primer). Each clone will be sequenced from each direction with M13F (-21) (25pmol; 2.5μL of 10μM stock) and M13R primers (25pmol; 2.5μL of 10μM stock) for a total of 10 sequencing reactions:
Clone #1 was selected from each of the screened clones.
A sterile pipette tip was used to inoculate 5mL of 1x LBAmp100 in a 15mL conical tube. The tubes were incubated O/N @ 37C on a rocker.
3mL of liquid culture was used as input for plasmid isolation with the QIAprep Spin Mini Kit (Qiagen) according to the manufacturer’s protocol.
1mL of each culture was combined with 1mL of 50% sterile glycerol (25% glycerol final concentration) and stored @ -80C with existing bacterial stocks.
Plasmid DNA was eluted with 50μL of Buffer EB and quantified on the Roberts Lab NanoDrop1000 (ThermoFisher) in order to have a rough idea of concentrations to submit for Sanger sequencing. A dye-based quantification will be performed after sequencing results are back in order to obtain a more accurate assessment for use in ISH and/or qPCR standard curve creation.
Quality (260/280 & 260/230 ratios) look great and yields are more than sufficient. Will prep samples for Sanger sequencing.
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.
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.
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.
The PCR products were separately ligated using The Original TA Cloning Kit (Invitrogen).
|REAGENT||SINGLE REACTION VOL (μL)||x 5.5|
|10x Ligase Buffer||1||5.5|
|T4 DNA Ligase||1||5.5|
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.
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.
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.
Both cloning reactions look great; lots of potential clones. Will screen nine from each via PCR for detection of our desired insert.
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.
Ample number of white colonies for all 4 sets of cloning reactions.
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.