Hellsgate and Ricky Boernke

[Peter.Waterhouse at csiro.au]

Dear Arabidopsis Researcher

We have been alerted to an email on the Arabidopsis network (from 
Ricky Boernke) about our Hellsgate vectors. We are confident that the 
proper Hellsgate8 vectors do work well, for us and many other labs. 
However, we may have identified the answer to the problem Ricky and 
some others are experiencing. There have been many hundreds of 
requests for the vectors, so we have had to make a number of batches 
of DNA. Last week we discovered that, unfortunately, at least one of 
the batches that we had sent out contained a Hellsgate precursor 
clone that was essentially the same as pHellsgate8 but which contains 
a direct repeat of the AttR sites rather than an inverted repeat (and 
consequently will not silence efficiently as it will not encode 
hairpin RNA). This plasmid contains a region:

35S promoter-AttR2-ccdB-AttR1-intron-AttR2-ccdB-AttR1-terminator.

It should be:

35S promoter-AttR1-ccdB-AttR2-intron-AttR2-ccdB-AttR1-terminator.

To make matters difficult, cutting with XhoI and/or XbaI (our usual 
diagnostic enzymes) gives the same size products for either plasmid.

However, digestion with PflMI (or an isoscizomer) or sequencing using 
the 35S primer (GGGATGACGCACAATCC) will differentiate between these 
two plasmids. Digestion with PflMI will give 9.0kb, 7.0kb and 1.4kb 
bands for the correct version and 8.2kb, 7.0kb and 2.3 for the 
incorrect version. Sequencing the correct pHellsgate8 with the 35S 
primer will give:



AGGAAGTTCA  TTTCATTTGG  AGAGGACACG  CTCGAGACAA GTTTGTACAA  AAAAGCTGAA 
CGAGAAACGT  AAAATGATAT  AAATATCAAT ATATTAAATT  AGATTTTGCA  TAAAAAACAG 
ACTACATAAT  ACTGTAAAAC



Another indicator of the incorrect plasmid version is that the likely 
recombination products after an LR reaction will not only be the 
direct repeat arrangement but also generation of a single gene 
fragment in the antisense orientation. The single antisense insert 
would be cut out by an XhoI-XbaI double digest but not by the single 
XhoI or XbaI digests that we recommend for checking recombinaton 
products.

We would like to apologize to anyone who has received this bad batch 
of Hellsgate 8. If you think you have a faulty plasmid, please 
contact us at <mailto:pi-bmta at csiro.au>pi-bmta at csiro.au and we will 
send a replacement.



We are a small research group with limited resources. This makes the 
large scale production and distribution of our plasmids difficult 
without severely affecting our research productivity.


Kind regards


Peter Waterhouse & Chris Helliwell