1 / General description / 1. pRTAC8 is a new transformation-competent artificial chromosome (TAC) vector suitable for constructing large-insert genomic library and transforming large DNA fragment in both monocot and dicot plants (Qu et al., 2003, Mol Breed 12: 297-308).
2. E.coli or Agrobacterium clones harboring pRTAC8 can be selected on LB or YEB containing 25 mg/L of kanamycin. For plant transformation, pRTAC8 can be transformed via both bombardment and Agrobacterium. In rice transformation, hygromycin in selection medium is 50 mg/L (Qu et al., 2003; Yin and Wang, 2000, Theor Appl Genet 100, 461-470).
2 / Components in pRTAC8 / 1. See Figure 1 as above.
2. pRTAC8 was derived from pYLTAC7 (see Note 3). Except for P35S, other components of pRTAC8 are the same as in pYLTAC7.
3 / pYLTAC7 / 1. pYLTAC7 is the original TAC vector used for large DNA transformation in Arabidopsis via Agrobacterium (Liu et al., 1999, Proc Natl Acad Sci USA 96: 6535-6540).
2. pYLTAC7 sequence is in GenBank AB020028 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=16874474 ).
4 / CaMV 35S promoter (P35S) / 1. The CaMV 35S promoter (Odell et al., 1985, Nature 313: 810-812) was used to construct “P35S::HPT::Tnos” in pRTAC8.
2. The 0.8-kb P35S was from pBI221. pBI221 sequence is in GenBank AF502128 (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=20454202 ).
pRTAC8 manuals
1. pRTAC8 was developed to construct rice TAC libraries that were used in cloning the rice Pi2 gene (Zhou et al., Molecular Plant-Microbe Interactions, 19:1216-1228). pRTAC8 was also used in rice transformation in the Pi9 gene research (Qu et al., Genetics, 172: 1901-1914) and in subcloning of genomic fragments in the Spl11 gene research (Zeng et al., Plant Cell, 16, 2795-2808).
2. The E.coli DH10B/TAC clones are very sensitive to kanamycin at the concentration of more than 25mg/L. To make sure kanamycin in LB does not go over 25 mg/L, we usually prepare a large stock (for example, 200 ml) of LB containing 20 mg/L of kanamycin.
3. We have successfully used the NotI, AscI, BamHI and HindIII unique sites in pRTAC8 for cloning large or small DNA fragments. Dephosphorylation of the digested vector is very important for obtaining high efficiency of positive clones. We usually perform dephosphorylation using HK Phosphatase (Epicentre Technologies, Wisconsin, USA). If HK Phosphatase is not available, you can try the shrimp alkaline phosphatase (Roche, Cat. # 1758250). It seems that calf intestinal alkaline phosphatase (CIAP) damages TAC vector termini.
4. SacB gene in the TAC vector is for selection against negative clones that were from vector self-ligation (Liu et al., 1999). We followed the 5%-sucrose selection procedure as described (Liu et al., 1999), but still got many negative clones when pTRAC8 was not efficiently dephosphorylated. The reason was unclear. It might be due to a high background in the SacB selection system. However, if vector dephosphorylation is successful, percentage of positive cloning can go over 90%. Based on our experience, the efficiency of TAC cloning primarily depends on how much the vector is dephosphorylated. We would suggest that you try dephosphorylation with different conditions (time is the major factor) and test the dephosphorylated vector in pilot ligations. Here is our protocol for pRTAC8 dephosphorylation.
1) Purify pRTAC8 using Qiagen large construct kit.
2) HindIII digestion (60ul of total volume): pRTAC8 (~ 0.7 μg/μl) 10 μl, 10x HindIII digestion buffer (or Roche 10x B buffer) 6 μl, HindIII (10 u/μl) 3-5 μl, double-distilled water 41-39 μl to bring to 60 μl; 37 ˚C/1.5 hrs.
3) Run the digested DNA in mini-gel (60 v/3 hr/ice bath), cut the band and column-purify the DNA.
4) HK-Phosphorylation: 60-80 μl of total volume, 1.0x TA buffer, CaCl2 to 50 mM, 2.5 μl HK Phosphatase, try 37 ˚C for 1.5 hrs, 2 hrs or 2.5 hrs, respectively.
5) DNA dialysis: float a Milliphore VSWP01300 filter membrane on sterile water (in Petri dish), pipette dephosphorylated DNA onto the membrane disc, and dialyze for 1 hr. Do not purify DNA using columns because phosphor-groups at the vector termini have been removed in the dephosphorylation step).
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The large DNA transformation vector pRTAC8 (The Wang Lab, Ohio State University)