dam Methyltransferase

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.

Protocol tips
S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.

Publication protocol

Immunodetection of methylated PCR products.
Portions (10 μl) of methylated and digested PCR products were run at 100 V for 45 min on a 10% polyacrylamide gel and then transferred to a positively charged membrane (Amersham Hybond-N+; GE Healthcare, Pittsburgh, PA) at 70 V for 30 min in 0.5× TBE buffer. The methylated DNA was detected by adding the anti-6m-A monoclonal antibody, at a 1:500 dilution for 30 min at room temperature. Biotin-labeled goat anti-mouse IgG (KPL, Inc., Gaithersburg, MD) was added to the membrane in a 1:500 dilution for 30 min at room temperature, and then 1 μg of phosphatase-conjugated streptavidin (KPL, Inc.) was added, followed by incubation at 30 min at room temperature. The reaction was developed with BCIP/NBT substrate (KPL, Inc.).

Detection of M. avium subsp. paratuberculosis in milk samples by immunocapture and IS900-PCR.
Pasteurized, cow whole-milk samples were purchased from a commercial supplier. The outside of the plastic bottle was cleaned with 100% ethanol, and the milk was aspirated with a sterile syringe and needle without opening the container. The M. avium subsp. paratuberculosis-free condition of the milk was confirmed by the lack of growth in 7H10 Middlebrook-OADC medium with mycobactin J and by DNA extraction by boiling, followed by PCR as described by Millar et al. (33). Samples analyzed using both methods were negative. Milk was spiked with different quantities of viable M. avium subsp. paratuberculosis cells (ATCC 19689). Some samples were not spiked with M. avium subsp. paratuberculosis (negative control). Samples were submitted to an immunocapture procedure for further PCR. Briefly, anti-mouse IgG Immuno-Magnetic Beads (IMB; Immunotech, France) were coated with a mouse anti-M. avium subsp. paratuberculosis polyclonal serum produced in our laboratory (anti-M. avium subsp. paratuberculosis-IMB). Next, 15 μl of the anti-M. avium subsp. paratuberculosis-IMB was added to 1 ml of milk that had been spiked with different quantities of whole M. avium subsp. paratuberculosis, incubated at room temperature for 15 min under agitation, and concentrated by magnetic separation. Preliminary experiments using milk spiked with 10-fold dilutions of M. avium subsp. paratuberculosis from 109 to 10 CFU/ml showed that 10 μl of anti-M. avium subsp. paratuberculosis-IMB can capture as few as 10 CFU in the lowest dilution and up to 103 in the higher dilutions. The presence of M. avium subsp. paratuberculosis in the magnetic beads fraction was determined by growth of colonies in 7H10 Middlebrook-OADC agar. No growth was detected in the supernatant of the fractions containing ≤103 CFU/ml (data not shown). The supernatant was removed, and the beads were washed three times with PBS for 2 min, resuspended in 100 μl of PBS (3 mM NaH2PO4, 7.5 mM Na2HPO4, 145 mM NaCl), and boiled at 100°C for 10 min to release bacterial DNA. Then, 10 μl of the supernatant was added to 90 μl of the PCR mix. PCR products were submitted to radiometric methylation and detected by gel electrophoresis or liquid scintillation. The whole procedure was repeated at least three times in three independent experiments.

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