Protocols[ edit ] DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through an agarose or acrylamide gel. The DNA is denatured and transferred from the gel to a solid support. The reason for transferring the DNA fragments to a solid support usually a nitrocellulose plate is that the DNA is inaccessible to DNA probes while embedded in the gel.
The technicians, students and guests working at the clinical DNA-diagnostic department. Responsibilities The technicians, students and guests using digestion, agarose gel electrophoresis and Southern Blotting for DNA-diagnostic analysis of specific diseases are responsible to perform these procedures according to this protocol.
After incubation all samples are tested for complete digestion. If the samples are digested completely, the procedure can be continued, otherwise an extra incubation step is performed. The procedure continues with the separation of the DNA fragments on an agarose gel.
An electric field is applied across the agarose gel on which the DNA samples are loaded. At neutral pH the DNA-fragments have a negative charge and will therefore migrate towards the anode positive charge.
The DNA-fragments are separated according to their size; the smaller fragments will migrate faster through the gel than the larger fragments.
After separation the DNA-fragments are transferred to a nylon membrane Southern blotting. The DNA in the gel is denatured with alkali-solution, subsequent capillary transport of the DNA fragments takes place from the gel to the nylon membrane, using the same alkali-solution.
The relative positions of the DNA-fragments are preserved during their transfer to the nylon membrane. After alkali-blotting the DNA-fragments are bound to the membrane and hybridization with a radio-labelled probe protocol ALG is used to detect specific fragments.
Durables balance, BP P, Sartorius combs, 24 or 28 teeth, 1. The other solutions are ready to use or made according to the protocols described here.
For one gel prepare half the amount alkali-solution 2 0. Dilute 20 ml of 10 x SLM to ml with Elga water. Store at room temperature spermidine see WFS04 ; 1. Complete the form before you start.
If necessary it is possible to take a multiple amount of DNA. The complete procedure takes 3 days. For pipetting water, spermidine and buffer there is no need to change tips NOTE: To get equal signals on your blot compared to normal blood-derived DNA samples, take twice the amount for the digestion if possible!
Depending on the capacity needed, two gel sizes are used: Disengage and slide the movable wall to the open end of the Gel Caster by turning and lifting the cam lever upward. Place the open end of the gel tray against the fixed wall of the Gel Caster.
Slide the movable wall against the edge of the gel tray. To seal the open tray ends, engage the cam lever by turning and pressing downward simultaneously, until resistance is felt the agarose should be allowed to cool to approximately 60oC before pouring into the gel tray cool the erlenmeyer under cold running tap water.
Pour the melted agarose into the tray and place the combs. Wait min until the gel has solidified carefully remove the combs from the solidified gel by lifting them straight up disengage the cam lever by turning and lifting upward.
Slide the movable wall away from the gel tray. Look critically at the photo to see whether all digestions are complete see Troubleshooting.
If not used on day 2, storage should be at oC in the freezer in the pre-PCR lab Agarose gel electrophoresis this part of the protocol is partly carried out at a pre-PCR lab preparation of the samples and partly at the post-PCR lab preparation of the gel, electrophoresis of the samples preparation of a 20 x 25 cm blot gel the Owl Scientific electrophoresis system is used and a gel is casted in a casting tray prepare a 0.
Load a maximum of 6 samples next to each other and skip one slot. Refresh the solution after 15 min NOTE: Mark the membrane with a black pen with the date and number of the gel if more then one gel is blotted.
Put Elga water in a tray, soak the membrane in it, pour off the Elga water and leave the membrane in alkali-solution for at least 5 min one GB paper same size as the gel one GB paper same size as the gel start to build the "blotting tower"; put a glass plate on top of 2 plastic trays 20 x 16 cm containing alkali-solution soak the two GB papers 35 x 19 cm or 46 x 19 cm in alkali-solution and put them on the glass plate.
Remove air bubbles by rolling with a glass pipet NOTE: Make a pile of about 10 cm high and put this on the blotting tower place a glass plate together with a weight of about gram on top NOTE: Neutralize the membrane in neutralisation buffer for min and dry it between filter paper check the membrane for correct blotting and possible introduction of air bubbles using a UV transilluminator cover it with Saran Wrap to protect the membrane.
Mark all empty lanes and if possible the slots on the DNA side of the membrane. Rinse the glass plates and trays with water to remove all alkali solution. Clean the table with water References  Maniatis, Sambrook, Fritsch: Molecular Cloning, 2nd edition 9.The power Point presentation contains 38 slides explaining about different steps involved in Southern Blotting such as DNA Isolation, Restriction digestion, Separation of DNA fragments by gel electrophoresis, denaturation of Double stranded DNA, transfer of fragments from gel to membrane (blotting), hybridization and detection by .
· Southern blotting, which is named after its inventor, Edwin Southern, is a common technique used in molecular biology to separate and characterize timberdesignmag.com is an effective way to identify a specific DNA pattern by the following timberdesignmag.com://timberdesignmag.com /DNA_recombinant_techniques/Southern_Blot.
· Analysis may involve a Southern blot to size and probe the PCR product, or DNA sequencing of the product to determine the precise sequence of bases. In contrast, multiplex PCRs begin with the simultaneous binding of many different primers (two for each of the loci)timberdesignmag.com Telomere length can be determined by a modification of Southern blotting in which the analysis of chromosome terminal restriction fragments (TRFs), as visualized with a StyI-digested λ DNA (or other appropriate unlabeled, digested DNA; see Support Protocol 1) Digest 1 µg DNA to completion for 4 hr to overnight at 37°C with ≥1 .
Invitrogen™ Anza™ 8 XhoI is a restriction enzyme that cuts DNA at this recognition site: C^TCGAG, completely digesting the DNA in 15 minutes at 37°C.
Analysis of nucleic acids Analysis of Nucleic Acids 1 Southern Blot Digest approximately 15µg of genomic DNA sample with appropriate restriction enzyme and.
· Selective blotting of restriction DNA fragments on nitrocellulose membranes at low salt concen-trations. Analysis of DNA. and immobilize the DNA by irradiating for the appropriate time (determined as described in UNIT A support protocol).timberdesignmag.com · The name western blot is a play on the eponymously-named Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Similarly, detection of RNA is termed northern blot and was developed by James Alwine, David Kemp, and George Stark at Stanford. Applications · Procedure · -D gel electrophoresis · See also · Referencestimberdesignmag.com · Characteristic examples of these techniques are Southern and northern blotting hybridization for the analysis of DNA and RNA, respectively In this chapter, the princlples, important practical aspects, and examples of apphcatlons of the Southern-blotting technique will be described, whereas northern blotting is dealt with in Chapter 9 in this volumetimberdesignmag.com