Total RNA (1 µg, originated from 2 × 106 CFU) was separated on a 6% denaturing polyacrylamide gel (Novex precast 6% TBE‐Urea polyacrylamide gel, Invitrogen™) run in a XCell SureLock™ Mini‐Cell (Invitrogen™) according to manufacturer's protocols. Prestained molecular weight markers (DynaMarker DM270; BioDynamics Laboratory Inc.) were separated alongside Mk total RNA and the 100‐bp hybridization probe fragment (see below), which was used as reference for maker size calibration. After electrophoresis, the gel was electroblotted onto a nylon membrane (Amersham Hybond N+; GE Healthcare Life Sciences) using a XCell SureLock™ Mini‐Cell Blot Module (Invitrogen™) in the XCell SureLock™ Mini‐Cell according to manufacturer's instructions and subsequently baked for 2 hr at 80°C. An Mk B11‐specific DIG‐labeled hybridization probe (100 bp; genome coordinates 2810109–2810208) was generated by PCR amplification using primers CB9c3 and CB8c and a PCR DIG‐labeled probe synthesis kit (F. Hoffmann‐La Roche, Ltd.) according to manufacturer's instructions. The probe was used for the hybridization analysis following reported protocols (Quadri et al., 1994 ; Sambrook & Russell, 2001 ) and visualized by chromogenic detection using a DIG‐labeled nucleic acid detection kit (F. Hoffmann‐La Roche, Ltd.) according to the DIG Application manual (F. Hoffmann‐La Roche, Ltd.).
Pcr dig labeled probe synthesis kit
Manufactured by Roche
The PCR DIG-labeled probe synthesis kit is a laboratory product that allows the synthesis of digoxigenin (DIG)-labeled DNA probes using the polymerase chain reaction (PCR) technique. The kit provides the necessary reagents and components to generate labeled probes for various applications, such as Southern blotting, Northern blotting, and in situ hybridization.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pcr dig labeled probe synthesis kit
1
Northern Blot Analysis of Bacterial RNA
2
Southern Blot Analysis of Transposon Insertions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of genomic DNA (0.5–1.0 µg) digested with SacII (an enzyme that does not cut within the Tn) were separated alongside a prestained DNA molecular weight marker (F. Hoffmann‐La Roche, Ltd.) by electrophoresis in a 0.7% agarose gel. After electrophoresis, the DNA on the gel was denatured and transferred onto a nylon membrane (Amersham Hybond N+; GE Healthcare Life Sciences) as reported (Quadri, Sailer, Roy, Vederas, & Stiles, 1994 ). The Tn‐specific 512‐bp DIG‐labeled DNA hybridization probe was generated by PCR amplification using primers GG1 and GG2 and a PCR DIG‐labeled probe synthesis kit (F. Hoffmann‐La Roche, Ltd.) according to manufacturer's instructions. The probe was used for the hybridization analysis following reported protocols (Quadri et al., 1994 ; Sambrook & Russell, 2001 ) and visualized by chromogenic detection using a DIG‐labeled DNA detection kit (F. Hoffmann‐La Roche, Ltd.) according to the DIG Application manual (F. Hoffmann‐La Roche, Ltd.).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!