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Frequently Asked Questions (FAQs)

 

1. Q: I am sorry to bother you, but several folks in my lab are having trouble with the pUNI system and I was wondering if you could give us any tips. We are following the in vitro recombination protocol given in the Elledge paper (Liu et al (1998) Current Biology) but are getting essentially no colonies after transformation with the recombination reaction. The very few colonies obtained don't look like either of the two parental plasmids or any combination thereof based on restriction analysis.

I don't think it's our Cre or our transformation protocol, as a control plasmid we purchased from NEB is showing recombination and efficient transformation. And we sequenced the lox sites in both the pUNI vector and the destination vector (pKYLX-myc9-loxP ) and they seem fine. pKYLX-myc9-loxP also seems fine by restriction digest (we got one aliquot of it from the ABRC that didn't give the expected digest pattern, but the second aliquot seems ok). Finally, people in the Callis lab report that they are getting very low frequency recombination using this system, although they are getting the desired plasmids after some effort.

How reliable is this system in your lab members' hands? And can you pass along any tips or suggestions?

   A: 1. When using commercial Cre recombinase (from Novagen), we need a much higher concentration (1ul/Rxn) than what they recommended (0.1 ul/Rxn). And even that, sometimes we only get less than 10 colonies. But what I get is always the correct. However, we routinely are using home-made GST-Cre, which we use at higher concentration, and this gives much better recombination frequency. Increase the amount of Cre does increase the recombination frequency.

2. The antibiotic selection for recombination is also very important. In this case, the recombinants should be Kan (50 ug/ml) and Tet (12.5 ug/ml) resistant. Usually, if you are using DH5a or Top10 strains, Kan selection is sufficient, because pUNI can not propagate in them.

3. The recombination events are reversible So we usually don't let the reaction go too long. 30 min in 37C is what I used. And I will do the transformation right away.

The UPS system is quite reliable in our hands. we have done numerous recombinations. The only time problems we have had getting the right clones was the antibiotics went bad. Other than that, we have never had any problems with the systesmt. Whenever we get colonies, they are always right. Occasionally, I get some duplex, but the frequency is low.

2. Q: I am trying to find a map of the vector pHB3-His6 (ABRC Stock# CD3-595). I did find out that it is a derivative of pET-15b (Novagen). However, I am unable to locate a map of pHB3-His. Can you help me in this?

I am interested in this vector because we want to use it for functional expression of some At cDNA clones (Uxxx) in E. coli. If you would suggest the use of another expression vector, suitable for expression (and His tagging) of UNI vectors in E.coli with the UPS method I would be happy to know.

   A: Click this link to get complete information (pdf) of UPS protocols and vectors.

3. Q: My lab has ordered last year a cDNA Salk pUni U67194 (At3g12100). I have started the sub-cloning in various vectors (and spent lots of money) then sent for sequencing. I've just found that this cDNA has a transposon inserted at around 250 bp from ATG (sequence matching the transposon sequence k17e7_5). I think this cDNA should be removed from your website or that you label it as containing a transposon. Your website specifies that you have sequenced all the salk clones.I don't understand why my clone has a transposon!? U Clone: It is a pUni clone that contains only the ORF of the R clone. It is sequenced and is error free?.

   A: In the cloning process we sequence single colonies that are innoculated into a 96-well plate for miniprep and glycerol stock. After I identify and fully sequence a clone, we use a robot to split the stock into 2 plates. One plate is used for a miniprep that is for sequence validation of the clone I am sending out. The other plate is robotically aliquotted into tubes that are sent to ABRC.The single end sequence of this clone used for validation matches the original sequence and there is no evidence of a transposon at 250bp when it is searched at NCBI to the nrdb. On our end U67194 was sent to ABRC intact. I am attaching the raw chromatogram data of the validation sequence for your review. It's most likely that the transposon hopped into your stock at an early stage of propagation of the vector in your laboratory. Its always a good idea to check any stock before spending a lot of effort/money on it. We realize resequencing is expensive and that is why we make the effort to produce error free clones. it would be useful for investigators to check (by PCR/restriction digestion) the size of the insertion to confirm that such an event (although relatively rare) has not taken place in their clone and as a confirmation that they have they are working with the correct material.

4. Q&A. I used the commercial cre protein as positive control, as you had suggested, and found the cause of my problems: As we have been working mainly with ampicillin-resistant vectors, we reduced the transformation protocols by omitting the incubation step in SOC medium after heat-shock. This worked perfectly with ampicillin-res plasmids, but not with kanamycin, as the cells had no time to express the neo gene before being killed by kan. When I introduced this 30 minutes incubation step, both commercial cre and cre produced based on your protocol started to work perfectly and efficiently.

I write this message to you, as I think some other people that have problems with UPS system (http://signal.salk.edu/cdna_FAQs.html) could use similar transformation protocols that me, and this could be the reason of their failure.


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