Recommendations
on how to handle
the clones you receive |
 |
Additional procedures and links can be found in the following document, included with shipments of requested BAC, PAC & fosmid clones.
The clones you receive from us are usually shipped in LB
agar stab culture format. Most (but not all) of the BAC or PAC clones are propagated in the DH10B E.coli host (either the original strain DH10B or the later T1-phage resistant version of DH10B). The bacteria harboring the plasmid (BAC, PAC, fosmid, cDNA or vector) have been inocculated into LB
agar (containing 12.5ug/ml chloramphenicol for BAC clones and
25 ug/ml kanamycin for PAC clones, or other antibiotics as appropriate for the specific clone). The LB agar is placed in a small tube and this arrangement is called a "stab-culture" by microbiologists. Stab cultures are typically shipped at ambient ("room") temperature and should be placed in a refridgerator (40C) upon arrival. The few days at room temperature don't harm the plasmid. However, please note that the culture has a finite
life at 4°C.
We recommend that you streak this culture
to single colonies on a LB agar plate with the appropriate antibiotic and
prepare a glycerol stock of the clones for long term storage
at -85°C. Please make sure also to confirm the content of the clones by your own favorite method (for instance PCR for a sequence expected to be unique for the clone). While our libraries have minimal well to well
contamination, some colonies may not contain the expected plasmids. In most cases this is due to a database mismatch (see our Q&A). Hence, it is important, before initiating any experiments,
to check 3-5 colonies (PCR, or diagnostic restriction digest). This to make sure
that the clone stock that we sent you is pure and represents the gene or sequence of interest.
How would you be able to know which E.coli host, cloning vector and antibiotic resitance apply to your clone? That is typically described on a webpage for the clone collection ("library") which contains the specific clone. For instance, the clone "RP11-163M23" is a member of the RP11 library (full name "RPCI-11"). In the left top corner of the current webpage, you will see a Google Site-Search Box). Type "RP11'" or "RPCI-11" to find other pages on our site with details related to this library. One of these pages contains a detailed description of the library and literature references: http://bacpacresources.org/hmale11.htm. Dish "163" contains 384 different BAC clones created in the vector pBACe3.6 and host DH10B. The requested clone is found at the intersection of row "M" and column "23". The vector defines the antibiotic resistance gene (nearly always chloramphenicol).
|
