Effect of Polybrene on the N-Terminal Sequencing of Peptides Bound to Polyvinylidene Difluoride Membranes
✍ Scribed by William E. Werner; Chris Grimley; Pau-Miau Yuan
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 167 KB
- Volume
- 237
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
bation, 10 ml 0.04 N NaOH was added. Cells of the control treatment were resuspended in 20 ml 0.02 N NaOH. Results, however, showed that zymolyase treatment did not improve but rather worsened PCR amplification.
(4) Amount of NaOH compatible with PCR: Under the present conditions, 0.02 N NaOH could be added up to one-tenth of the total PCR reaction volume FIG. . PCR of 12 independent yeast colonies harboring a yeast/E. without obvious deterioration in amplification. Alcoli vector with various Arabidopsis thaliana cDNA inserts. Yeast ternatively, a larger volume of cells could be used if cells from single colonies were suspended in 10 ml 0.02 N NaOH and 2 ml was used in 30 ml PCR. The insert was amplified with two vector resuspended in 0.01 N NaOH.
primers for 30 cycles. The reaction mixture (7 ml) was electrophoresed in 2% agarose gel. S, 1-kb DNA ladder; 1-12, PCR of 12 yeast colo-Although the present method was effective for amnies. plifying sequence from low-copy plasmids, it would be still more useful if it was equally effective with genomic sequences. Thus, tests were performed to REFERENCES amplify fragments of two single-copy genes, CDC28 and CLN1. As in the case of plasmids, clear amplifi-1. Gu ¨ssow, D., and Clackson, T. (1989) Nucleic Acids Res. 17, 4000. 2. Huxley, C., Green, E. D., and Dunham, I. (1989) Trends Genet. cation was observed with cells in 0.02 or 0.04 N 6, 236. NaOH, while cells in H 2 O alone only gave a weak 3. Sandhu, G. S., Precup, J. W., and Kline, B. C. (1989) BioTechamplification (for the smaller CDC28) or no amplifiniques 7, 689-690.
cation (for larger CLN1) (Fig. ).