Frequently Asked Questions about Clones

Antibiotic Usage

Transformations

Obtaining information on cDNAs and ESTs

Drosophila Species cDNA

Drosophila Species Fosmids

FMO clones

UFO clones


Antibiotic Usage


Chloramphenicol - what concentration should I use?

Chloramphenicol concentration requirements in plates versus media is different. At the DGRC, we make a 34 mg/ml stock solution in 100% ethanol (store stock at -20). Then for liquid cultures, add 2 microliters of stock to for every 1 ml of LB liquid culture (you can also get by with 1 microliter per ml if necessary). For plates, add 1 ml of stock chloramphenicol solution for every 1 Liter of media for plates.


Transformation


Can I use electorporation on the Whatman discs?

Yes - Whatman has documented that this does work. We have not tried it at the DGRC but we have received the following information from a colleague who succesfully used electroporation: After rinsing with the TE for a few seconds he added electrocompetent XLI Blue cells (the lab's homemade) to the disc and incubated on ice for one minute. He then transferred the cells to a cuvette and electroporated. He plated the entire transformation on amp plates and obtained around 5 x 10^3 colonies.

Can I use glycogen precipitation?

We know of one person who tried a glycogen precipitation and centrifugation protocol as an alternative for recovering DNA from discs and did not have great success. We do not recommend trying this procedure as a means of recovering.


Obtaining information on cDNAs and ESTs


Where can I order EK or Exelxis ESTs I've seen listed in FlyBase?

Information we obtain from Exelixis: The Exelixis cDNA libraries were random-primed, rather than poly-dT primed, and so unlike the BDGP cDNAs, they were not full-length clones. Exelixis provided BDGP and Flybase with their EST sequences, but the clones were discarded because a majority were redundant with the BDGP EST collection.

Where can I find out more about my clones (restrictions sites, sequence, etc.)?

First, go to: http://www.fruitfly.org/EST/faq.html#cdna-1 This takes you to a page on the BDGP website. Look through this page and find the description of the library that your clone came from (the first two letters of your clone ID). In the paragraph describing the how the library was made, you should find the vector highlighted. Click on it and it will take you to a map and sequence information. To find out how much of your gene is cloned into the vector, go to the Flybase record we provide for the clone (search for your clone on our website and use the hyperlink we provide on the clone's page retrieved from the search. If you blast the sequence found in the Flybase record against the known or hypothesized full length cDNA of your gene (can also be found through FlyBase, then you'll know if your clone is full-length or will at least have an idea how much of the cDNA you have. After playing around on these pages, if you still have some questions, email us.

How do I find maps and sequence information on the Gateway clones (Terence Murphy collection)?

To locate this information, please visit
http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html

The GOLD clone I found on the BDGP website is not in the DGRC GOLD collection catalog.

There is a small subset of GOLD clones that have not been shipped to us yet. These are clones that BDGP has on partial 96-well format plates. Once the plate is complete, it will be sent the DGRC. We do not have a time estimate for receiving these clones. These GOLD clones can be found in the DGC or EST collections - please enter your clone ID through the DGC or EST collection page and place your order.

How do I order BAC clones?

The DGRC does not have BAC clones. BAC clones generated by BDGP can be obtained from the BACPAC Resource Center. Information can be found at http://bacpac.chori.org/.

Please note that the BDGP and BACPAC Resource Center have slightly different nomenclature systems for the BAC clones. A clone called "BACR48M07" by the BDGP should be referred to as "RP98-48M7" in communications with BACPAC.

What vector was used to make the IP clones and what antibiotic do I need to use for the IP clones?

The IP clones were made using pOT2. This vector is chloramphenicol resistant.


Drosophila Species cDNA


How were the species cDNAs cloned?

Species cDNAs were cloned into pAGEN-1, into SmaI (5' end) and Not I (3' end). Download a PDF of the pAGEN-1 map.

What was the size selection when making the libraries?

Size selection of cDNA was > 1.2 kb. Clones may or may not be full length.

Were there any other criteria used to select clones?

No.

What primers were used for sequencing?

M13 reverse ( for 5' end) and 2 bp clamp primer (for 3' end).
The 2 bp clamp primer sequence is 5' TTTTTTTTTTTTTTVN 3'- number of T's is variable, usually about 15, V = A, C or G; N = A, C, G or T. It is designed to bind at the poly A tail-cDNA junction.


Drosophila Species Fosmids


What vector was used to clone the fosmids?

pCC1Fos vector from Epicentre Sequence can be found on Epicentre's website.

How was the genomic DNA cloned in (linkers, sites, etc.)?

Blunt cloned into Eco72 I site.

What it the typical insert size?

38-42 kb.

What primers were used for sequencing?

FosF3 5' AGC TCG GTA CCC GGG GAT 3'
FosR3 5' AGG TCG ACT CTA GAG GAT 3'


FMO clones


What is the resistance in the FMO clones?

The FMO clones are both an ampicillin and chloramphenicol resistant.

Where is the FLAG tag in the FMO clones?

The FLAG-HA tag is further downstream of the ORF, beyond the chloramphenicol resistance marker. The HN-Tag almost immediately follows the ORF, and is expressed in prokaryotic systems. Upstream of the HN-Tag, there is a splice donor, which allows splicing to the FLAG-HA tag

The tag you see if you visit the BDGP community query clone report web page is that of the 6xHN tag. Keep in mind that the sequence on this page is just the 5' end verification read (to confirm transfer of the ORF into the vector).

However, there is also a splice donor immediately upstream of the 6xHN tag, and a splice acceptor downstream, beyond the chloramphenicol resistance marker. See this page for a schematic:
http://fruitfly.org/EST/proteomics.shtml

To view the predicted vector map of your clone, you can follow this link:
http://fruitfly.org/EST/proteomics.shtml#seq_generator

Just type in the name of your clone, e.g. UFO04389, then follow the subsequent link.


UFO Clones


What is the resistance in the UFO clones?

The UFO clones are both an ampicillin and chloramphenicol resistant.


Citing the DGRC

When publishing experiments using materials obtained from the DGRC please cite the Drosophila Genomics Resource Center, supported by NIH grant 2P40OD010949-10A1, in the acknowledgments. Your cooperation helps us when we need to renew our grant.