Improved in Vivo Dimethyl Sulfate Footprinting Using AlkA Protein: DNA–Protein Interactions at the Mouse H19 Gene Promoter in Primary Embryo Fibroblasts
✍ Scribed by Piroska E. Szabó; Gerd P. Pfeifer; Feng Miao; Timothy R. O'Connor; Jeffrey R. Mann
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 147 KB
- Volume
- 283
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
6000, Fluka), 100 mM CaCl 2 , 0.6 M KCl, 10 mM Tris-HCl (Sigma), pH 7.5) were added and the mixture was incubated on ice for 20 min. One milliliter of room temperature Solution E was then added and the incubation proceeded for an additional 20 min at room temperature. Aliquots of the transformation reaction were then mixed with 20 ml of recovery agar (equilibrated to 46°C), poured into empty petri dishes, and incubated for 3-4 days at 32°C.
The data in Fig. 1 compare the transformation efficiencies obtained in the presence and absence of DTT with all three species of DNA and strain GR5. Inclusion of 1 mM DTT in the protoplasting medium and Solution D enhances the efficiency by 2-to 13-fold on average, with differences in individual experiments as high as 60-fold. In all experiments more transformants were obtained in the presence of DTT than without. The mean numbers of transformants obtained per microgram DNA for all experiments described in Fig. 1 were 42.1 (pAL5, DTT), 3.3 (pAL5, no DTT), 179.0 (pXX1, DTT), 98.0 (pXX1, no DTT), 878.3 (pAID, DTT), and 198.4 (pAID, no DTT). Similar increases in efficiency were also noted for the poorly conidiating strains AO1 and DW61. One possible explanation for the increased efficiency was the improved viability of the protoplasts in the presence of DTT. However, the protoplast survival rates with or without DTT on nonselective medium (recovery agar plus uracil and uridine) were identical. Therefore, the effect must be due to an enhancement of the transformation mechanism itself.
Additional experiments to determine the optimal concentration of DTT demonstrated that transforming strain GR5 in the presence of 0.1 or 10 mM DTT did not provide a marked benefit. Using pXX1 and 0.1 mM DTT, the efficiency did improve by 1.5-fold, although with 10 mM DTT no more transformants were observed than for the control that lacked DTT. With the replicating plasmid pAID, values were unchanged from the control in the presence 0.1 mM DTT but reduced by almost half with 10 mM DTT. Therefore, a DTT concentration of 1 mM appears optimal for providing the highest efficiencies with a variety of plasmids for this strain.
We have now used this modification to successfully increase the number of colonies obtained with lowfrequency transformations including a linear fragment for deletion of a conditionally lethal gene and direct transformation of ligation mixes. We anticipate that adding DTT during the preparation of protoplasts will provide a useful enhancement of the transformation protocol for other A. nidulans strains and filamentous fungi in numerous applications.