TECHNICAL MANUAL

Dharmacon cDNA Clones

Product description
Clones are provided as Escherichia coli cultures in LB broth with 8% glycerol,
an inert growth indicator, and the appropriate antibiotic at the concentration
indicated in Table 1.

(ts.dharmacon@horizondiscovery.com, 800 235 9880) with your orderinformation and sequencing data and we will supply the correct clone
if possible.
Making a stock culture

Table 1. Cap color code

Color

Antibiotic

Concentration

Red

Carbenicillin*

100 μg/mL

Black

Chloramphenicol

25 μg/mL

GreenKanamycin

25 μg/mL*Ampicillin can also be used at the same concentration.

Once the clone has been streak isolated and the identity of the clone has
been confirmed, we recommend making a stock of the pure culture. Grow
the pure culture in LB broth with the appropriate antibiotic Transfer 920 μLof culture into a polypropylene tube and add 80 μL sterile glycerol to makean 8% glycerol freezing solution. Vortex the culture to evenly mix the glycerol
throughout the culture. The culture can be stored indefinitely at -80 °C.

Shipping and storage
Individual clones are shipped at room temperature and may be stored for
up to one week at +4 °C. Orders of greater than 50 individual clones will
be arrayed in a 96 well format. Plates are shipped on dry ice and should bestored at -80 °C. Clones may be stored indefinitely at -80 °C.

Plate replication protocol

Clone verification
All DNA sequences and annotations have been submitted to GenBank by
the supplier at the time of library creation (For example, the IMAGEConsortium), but have not been independently verified. Information about aparticular clone can be found by entering the Clone ID number or accession
number into search box at the top right of the webpage at (dharmacon.com).
Further verification can be performed by using BLAST or other bioinformatic
tools (blast.ncbi.nlm.nih.gov/Blast.cgi). If you have difficulty finding yourclone please contact Technical Support (ts.dharmacon@horizondiscovery.
com, 800 235 9880) for help.

Prepare source plates• Remove the foil seals from the source plates. Removing the seals while
the source plates are frozen will minimize cross-contamination.
• Thaw the source plate.

•Gently remove the replicator from the source plate and carefully place
the replicator into the target plate.

Protocol•Gently move the replicator back and forth in the target plate totransfer cells.

•Discard the replicator.

Sequence verifying individual cDNA identity
To ensure you are working with a clone is derived from a single isolate,streak out and isolate at least 3 individual colonies for sequencing.
The sequencing primers listed under the details tab for each clone.
In cases of incorrect clone identification please contact Technical Support

Prepare target plates• Dispense ~ 160 μL of sterile LB medium into 96-well microtiterplates. The LB should be supplemented with 8% glycerol andthe appropriate antibiotic.

Replicate• Gently place a disposable replicator into the thawed source plate andgently move the replicator around inside the well to mix the culture.
Make sure to scrape the bottom of the well.

Table 2. Materials for plate replication

Item

Vendor

2x LB-Lennox Broth (low salt)

Fisher Scientific

BP1427500

GlycerolFisher Scientific™

BP2291

Carbenicillin or Ampicillin

Fisher Scientific

BP2648-5 or BP1760-5

Chloramphenicol

Fisher Scientific™

BP904-100••••

Cat #™

™

Kanamycin

Fisher ScientificBP906-5

96-well microplates

Nunc™

12-565-363

Aluminum seals

Nunc™

12-565-475

Disposable replicators

Fisher Scientific™

NC9584102™

Seal the source plates, being mindful to avoid cross contamination.
Repeat this process until all plates have been replicated.
Return the source plates to the -80 °C freezer.
Place the inoculated target plates in a 37 °C incubator. Incubate the plates for 12-24 hours.

FAQS/troubleshooting
For additional information, please contact Technical Support (ts.dharmacon@horizondiscovery.com, 800 235 9880).
Questions

Answers

Where can I find acomplete vector mapof my cDNA clone?

We were not provided with complete maps for the clones in this collection, but the information available online can be used to create amap. You can find the details for your clone by clicking on the [+] next to your clone’s description. The “Details” tab includes a hyperlink
to the plasmid sequence as well as an indication of the 5’ and 3’ cloning restriction sites. The “Sequence” tab includes a link the clone’s
insert sequence.

What is the appropriate
antibiotic to use with mycDNA clone?

Clones are provided as E. coli cultures in LB broth with 8% glycerol, an inert growth indicator, and the appropriate antibiotic at theconcentration indicated either from our website or using the tube cap color as an identifier. The cap color codes are below:
Red - Ampicillin (100µg/ml)
Black - Chloramphenicol (25µg/ml)
Green - Kanamycin (25µg/ml)

Why is my bacterial
glycerol stock yellow?

Some of our E. coli cultures are grown with an inert growth indicator, phenol red. This indicator is red at pH 8.2 and above and begins toturn orange and then yellow as the pH drops. At pH 6.8 the indicator is yellow. As the E coli are growing they give off
CO2 which decreases the pH of the broth and changes the color of the indicator from red to yellow.
First ensure that you are using the correct antibiotic and concentration for your clone. You can find this information as described in theClone Details section of this document.

What would you suggest
I do if my cDNA clone is
not growing?

Additional strategies that may be used to spur growth include:• Try inoculating from thawed source tube rather than frozen.
• Gently mix the source tube by inversion (with the lid on) to ensure the cells are not settled in the bottom before theinoculum is taken.
• Try using broth culture instead of plate culture to jumpstart growth.
• Use shaking during growth of the broth culture.
• Try increasing the inoculum (i.e. 10ul inoculum into 2ml broth).
• Try spinning the tube to pellet the cells and take a portion of the pellet to inoculate a broth culture.

For additional Frequently Asked Questions (FAQs), please visit here.

Good Faith Agreement/License Agreements
For questions regarding license agreements please visit: http://dharmacon.horizondiscovery.com/uploadedFiles/dharmacon-product-terms-and-conditions.pdf
and/or contact Technical Support (ts.dharmacon@horizondiscovery.com, 800 235 9880).

t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f + 44 (0)1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Fisher Scientific and Nunc are trademarks of Thermo Fisher Scientific, Inc.
©2018 Horizon Discovery Group Company—All rights reserved. First published March 2018. UK Registered Head Office: Building 8100, Cambridge
Research Park, Cambridge, CB25 9TL, United Kingdom.

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If you have any questions