Two protocols are given here. The first one is used for BAC library screening on filters , although is also suitable for Transfer hybridizations. PREPARATION OF HYBRIDIZATION SOLUTION Prehybridization solution Final
* Boiled for 10 min then cooled on ice. Then added to the solution at 65℃. Prehybridize at 65℃ for 2 to 8 hours, then pour of prehybridization solution; it can be stored at 4C and reused over a week or so. Hybridization solution Final
* Boiled for 10 min then cooled on ice. Then add to the solution at 65℃. Add hybridization solution of membranes and rotated for 30 min to 4 hr before addition of labelled probe. The hybridization solution is identical to the pre-hybridization solution except for the inclusion of dextran sulphate. This polysaccharide increases the effective probe concentration in the hybridization solution by displacing probe from the volume occupied by the polymer. DNA Labelling procedures Follow a protocol like this or use a kit - BioLine HYPER-Prime, GibcoBRL (and previously Roche but we find their products do not have the reproducibility and robustness of the former Boehringer) are used in our laboratory.
Filter washing: preparation of washing solution
|
Preparation of Denhardts Solution (30ml) Preparation of 50% dextran sulphate (10ml) 5g Dextran sulphate Pat Heslop-Harrison, Karine Alix, Saadiah Jamli University of Leicester LE1 7RH Protocol 2. From Sybille Kubis, Maria Madon and Pat Heslop-Harrison. SOUTHERN HYBRIDIZATION PROTOCOL Prepare prehyb- and hybridization solution which consists of the following components: 1 mM EDTA pH 8.0
For prehybridization:
Note: The prehyb- and hybridization solution is the same. 60 ℃ for lower stringency Southern hybridization. 65℃ for higher stringency Southern hybridization. For probe labelling: Use RadPrime DNA Labeling System (Invitrogen)
20 µ l 2.5x Random Primers Solution 7 µ l distilled water
|
Washing of membranes:
rinse membranes :- i. Once with ~12.5 mls 2xSSC/0.1% SDS at room temperature. Drain the solution carefully into the sink outlet. ii. Then twice with ~12.5 mls 2xSSC/0.1%SDS at 60 ℃for 30' by rotating in the hybridization oven. If hybridization were done at 65℃, rinse membranes :- i. Once with ~12.5 mls 2xSSC/0.1%SDS at room temperature. Drain the solution carefully into the sink outlet. ii. Twice with ~12.5 mls 2xSSC/0.1%SDS for 30' at 65 ℃. iii. Once with ~12.5 mls 1xSSC/0.1% SDS for 15' at 65 ℃. Note: Washings are done in the Hybaid tube itself. Preparations of washing solutions: Autoradiography:
|
→如果您认为本词条还有待完善,请 编辑词条
词条内容仅供参考,如果您需要解决具体问题
(尤其在法律、医学等领域),建议您咨询相关领域专业人士。
0