Clones, Vectors and Strains
Clones, vectors and host strains are shipped as bacterial stabs or yeast slants in a small vial of agar media containing the appropriate selective agent. We recommend streaking out these stocks on an agar plate containing the appropriate selective agent as soon as possible after you receive your order.
If a stock does not grow when struck out from the stab or slant you received, check that you have used the correct selective agent at an appropriate concentration. We recommend using no more than 25 µg/ml kanamycin for full length cDNA clones in pUNI and Gateway® vectors and for BAC clones from the P1 and TAC libraries. If you continue to see no growth, order the stock again through TAIR and add a comment to your order describing the problem. A replacement stock will be shipped free of charge.
Select several colonies from the agar plate on which you struck out the stock from a stab or slant culture, to inoculate cultures for verification of the stock you have received.
Use an appropriate method such as restriction analysis, PCR amplification, or sequencing of the insert to verify stocks.
Once you have obtained a verified culture, make a glycerol stock or isolate a sample of plasmid DNA for long term storage. This is important as stabs received from ABRC may have spent several weeks in shipping and were not stored under ideal conditions during this time.
If you are unable to verify a stock, check that you have used the correct selective agent at an appropriate concentration and that you have analyzed several colonies derived from the original culture. If possible, use more than one method of verification, such as PCR amplification and restriction analysis. Please report continuing problems to ABRC as soon as possible. In most cases you will be instructed to order the stock again through TAIR and we will provide you with a culture derived from an archived stock that has undergone minimal handling at ABRC. It is not possible for us to verify every stock before distribution to the community, so we rely on feedback from researchers to identify problems with stocks. If you sequence a stock, please submit the sequence to ABRC so that we can attach it to our records of the stock.
Some common problems with clones
- A cDNA clone is not full length or is not the same size as the available sequence of the clone. If the clone is an EST it has only been end sequenced. In many cases it is not possible to determine whether the clone is full length, or what the exact size of the insert should be as it has not been fully sequenced. Most EST clones are longer than the available sequence, but many do not represent a full length cDNA. If the clone is represented as a full length cDNA in TAIR the gene may be annotated incorrectly or the annotation may have changed since the clone was submitted.
- A full length pUNI clone appears to lack an insert. We have documented some cases of these clones losing their inserts, but they appear to be relatively rare. Restriction analysis using SfiI is not always a reliable method of dropping out the insert because the SfiI sites may become methylated. We recommend using HindIII and EcoRI. In cases where the insert really is lost we find that we can maximize the chances of finding and insert by using a culture that is as fresh as possible and checking several colonies.
- A restriction digest or end sequencing indicates that the clone is wrong. For clones such as BACs that were received and have been maintained in 384 well microtiter plates, or clones form high throughput collections, there is always some possibility that the stock we provided was selected from the wrong well, or that cross contamination between wells occurred. In some cases, checking several colonies will yield the correct clone. In others we are able to supply a new stock derived from an archived stock that represents the correct clone. In some cases we are not able to identify the correct clone and we will provide an alternative stock if possible or refund or cancel the charges for your order.