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Southern杂交
One important thing for transfer:the weight of the object resting on top of the blotting apparatus should not exceed the weight equal to a 500 mL, half full bottle, for it will give your (nitrocellulose) membrane blotches. (Contributed by Alex Ramirez )
· Southerns, Westerns, and Northerns: Molecular Cloning Techniques (Brian White, MIT)
Basic theory, definition and procedures with illustration
· Southern Blot Preparation (gopher://iubio.bio.indiana.edu/)
· Southern Blot (Crawford Lab)
· Southern Blotting (Gimila Lab)
· Southern Blotting (Corbett Lab)
- Southern Transfer to Nylon Membranes (Donis Keller Lab)
Southern transfer is the classic method used to move DNA (usually in the form of restriction fragments) from agarose gels to a solid support, e. g. nylon membrane. This protocol is designed for transferring DNA for RFLP analysis. The optimum size range of DNA fragments detected this way is 2 - 25 kb.
- Southern Transfer to Immobilon-N (Donis Keller Lab)
Immobilon- N is a non-nylon membrane that can be used very much like Zetabind in Southern transfers. Because it is extremely hydrophobic when dry, the membrane must first be prewetted in alcohol prior to use.
- Southern Transfer to Nylon Membrane in NaOH (Donis Keller Lab)
The NaOH transfer may be a more efficient transfer method for larger sized fragments. It is the method of choice for pulsed-field gels containing fragments >= 20 kb.
- Primer Extension Labeling of DNA in Low Melt Agarose (Donis Keller Lab)
This procedure allows a restriction fragment(s) to be labeled directly from a gel slice, without the associated losses of DNA usually seen with gel purification methods.
- Labeling DNA with 32P by Primer Extension (Donis Keller Lab)
Double stranded DNA is heat-denatured and random hexanucleotide primers are attached to the single strand. The synthesis is carried out using Klenow fragment of DNA polymerase I incorporating 32P-dCTP at the same time.
- Prehybridization of Repeat-Containing Clones with Total Human DNA (Donis Keller Lab)
To reduce the lane background due to the repetitive sequences, large amount of unlabeled human DNA is added to the labeled probes immediately before the denaturing and hybridizing steps...
- Hybridization to Southern Blots(Donis Keller Lab)
To hybridize radioactively labeled DNA probes to Southern blots containing human DNA to detect Restriction Fragment Length Polymorphosims (RFLPs). This method can also be used for most other applications (e.g., slot blots, zoo blots, yeast YAC blots etc.).
· Southern blotting (UMBC)
· Southern Blot (Bowtell Lab)
· Southern Blot (PMCI Research)
· Southern Blot Hybridization(Tyner Lab)
Procedures, recipes, and stripping for rehybridizatioon...
· Southern Hybridization (ASIRC)
· Southern Transfer and Hybridizatioin (Gottschling Lab)
· Analysis of Genomic DNA by Southern Hybridization(Jun-Ryul Huh)
Great protocol for Southern including Probe preparation, filter preparation, filter suppression (pre-hybridization), probe suppression, washing and exposure
· Alkaline Southern Blotting (Kim Marshall, Dendrome)
· Southern blot-chemiluminescent detection, using Lumigen or Lumiphos (NWFSC)
Non-radioavtive detection
· Southern blot-chemiluminescent detection, using CSPD (NWFSC)
· UV Cross-Linking (gopher://iubio.bio.indiana.edu/)
- Making Combination Markers for Size Standards on Southern Blots(Donis Keller Lab)
Digesting lambda DNA (BRL) with a specific restriction enzyme will result in lambda DNA fragments of known size. Three separate lambda digests (BglII, BstEII, and XhoI) will give 23 fragments with a molecular weight range of 60 bp to 33,498 bp (see illustration below). The three digests are pooled, diluted, and used as molecular weight size standards for Southern transfers.
- Preparation of Sonicated Human DNA(Donis Keller Lab)
To break up high molecular weight human placental DNA into fragment sizes of 500 bp or less which can be used as competitor DNA in Southern and in situ hybridizations. This method can also be used for sonicating salmon sperm DNA.
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