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RNA实验方法-2

2019.5.18

13. Prehybridization mix (add in order listed at 5 mL/100 cm² membrane)

Conc.	5 mL	10 mL	15 mL	x mL	Stocks
5x	500 礚	1000 礚	1500 礚		50x Denhardt誷
5x	1 mL	2 mL	3 mL		20x SSC
50 mM	250 礚	500 礚	750 礚		1 M NaPO₄, pH 6.7
50%	2.5 mL	5 mL	7.5 mL		deionized formamide
250 礸/mL	250 礚	500 礚	750 礚		5 mg/mL sheared salmon sperm DNA
1%	100 礚	200 礚	300 礚		50% dextran sulfate MW 5000
	350 礚	700 礚	1050 礚		Type I water
0.1%	50 礚	100 礚	150 礚		10% SDS
	5 mL	10 mL	15 mL	x mL

5x Denhardt誷, 5x SSC, 50 mM NaPO₄, pH 6.7, 50% deionized formamide250 礸/mL sheared salmon sperm DNA, 1% dextran sulfate MW 5000 and 0.1% SDS

Prep the hyb mix and prehyb mix fresh each time. The hyb mix can sit at room temperature during the 3 hour prehyb step.

14. Hybridization mix (add in order listed at 5 mL/100 cm² membrane)

Conc.	5 mL	10 mL	15 mL	x mL	Stocks
1x	100 礚	200 礚	300 礚		50x Denhardt誷
5x	1 mL	2 mL	3 mL		20x SSC
20 mM	100 礚	200 礚	300 礚		1 M NaPO₄, pH 6.7
50%	2.5 mL	5 mL	7.5 mL		deionized formamide
100 礸/mL	100 礚	200 礚	300 礚		5 mg/mL sheared salmon sperm DNA
5%	500 礚	1000 礚	1500 礚		50% dextran sulfate MW 5000
	650 礚	1300 礚	1950 礚		Type I water
0.1%	50 礚	100 礚	150 礚		10% SDS
	5 mL	10 mL	15 mL	x mL

14. Hybridization mix (add in order listed at 5 mL/100 cm² membrane):

1x Denhardt誷, 5x SSC, 20 mM NaPO₄, pH 6.7, 50% deionized formamide, 100 礸/mL sheared salmon sperm DNA, 5% dextran sulfate MW 5000, and 0.1% SDS

15. Wash square Pyrex dish that will fit in shaking waterbath. Soak in 3% hydrogen peroxide or 0.1% DEPC for 10 min.

16. High Stringency Washes 1, 2 and 3

Wash 1 2x SSC	Wash 2 1x SSC	Wash 3 0.5x SSC
80 mL	40 mL	20 mL	25x SSC, pH 7.0
910 mL	940 mL	970 mL	Type I water
10 mL	10 mL	10 mL	10% SDS
1000 mL	1000 mL	1000 mL

Wash 1 2x SSC: 80 mL 25x SSC, pH 7.0, 910 mL Type I water, 10 mL 10% SDS

Wash 2 1x SSC: 40 mL 25x SSC, pH 7.0, 940 mL Type I water, 10 mL 10% SDS

Wash 3 0.5x SSC: 20 mL 25x SSC, pH 7.0, 970 mL Type I water, 10 mL 10% SDS

17. Discard the probe and hybridization solution in ³²P liquid waste. Rinse the bag with approximately 30 mL wash 1 and discard the wash in ³²P liquid waste. Discard the 0.1% DEPC in the tray and add approximately 100 mL wash 1 to the dish. Transfer the membrane to the dish and cover with Saran. Shake gently at room temperature for 10 min. Discard wash in ³²P waste. Microwave the remaining wash for 1 min to prewarm slightly and add to the Pyrex dish containing the probed membrane. Incubate at 65 for 30 min. Washes may be performed in the hybridization bag, but may result in higher background. Discard wash 1 and add 1000 mL prewarmed wash 2 and incubate at 65 for 30 min. Discard wash 2 and add 1000 mL prewarmed wash 3 and incubate at 65 for 30 min.

18. Air dry the filter on 3MM paper and wrap with Saran. Expose the membrane as desired.

Reprobing

Do not let the membrane dry out if reprobing is required. Place the blot in a clear plastic sheet protector and seal it closed with tape for chemiluminescent exposure. For storage, seal the blot in a water-tight seal-a-meal pouch and store at ?0. Note that only SaranWrap is water-tight. Other plastic wraps are water-permeable and allow blots to dry out (and allow water from wet gels to contact autoradiography film, causing black spots).

Charge

Neutral membranes have an equal and uniform distribution of charges. Positively charged membranes have a preponderance of positive charges across the matrix. They will hold the same amount of nucleic acid. Positively charged membranes have stronger retention: the target remains bound longer. UV crosslinking forms a covalent bond between nucleic acids and the positive charges of the membrane. Fixation by baking creates a hydrophobic interaction between the nucleic acids and the membrane. If stripping and reprobing is required, it is better to UV crosslink to a positively charged membrane in order to preserve the integrity of the target during the stripping step.

Sidedness

Quality membranes do not have a correct side. The charges are equally distributed on both sides.

Porosity

Nominally, 0.22 祄 pore membranes have greater surface area than 0.45 祄 membranes. They also require better blocking. Consistent pore size is critical. For Northern blots of small RNAs (~ 100 nucleotides), 0.1 祄 membranes are recommended.

Background

Particulates in unfiltered buffer, skin oils, nonspecific antibody binding, addition of too much probe, inadequate post-hybridization washing, improper wash temperature, and other factors can create background fog or spots on membranes.

Blocking

Blocking can reduce background. Non-specific proteins block certain electrostatic interactions which may attract proteins randomly. Milk proteins, BSA, or commercial blocking agents are typically used.

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Ribonuclease Protection Assay

contributed by James McCaughern-Carucci, Yale University

Most RNase Protection protocols require an overnight hyb with numerous subsequent clean-up steps. This method requires a maximum hyb of four hours, and the clean-up steps are the barest minimum, yet still produce nice images. It is strongly recommended to titer RNase concentrations with probes prior to running experiments...some require more RNase than others.

Part I: In Vitro Transcription

In a sterile 1.5ml microfuge tube, combine the following:
("ul"= microliter; all reagents obtained from Promega)

4 ul 5x Transcription Buffer
2 ul 0.1M DTT
4 ul 2.5mM NTP's (A, C, G)
0.8 ul RNasin RNase Inhibitor (25U/ul) (Promega)
2.4 ul of 100uM cold UTP (Note: Use 1mM UTP for loading control probes e.g. B-actin)
1 ul of 1 ug/ul linearized DNA template
5 ul of 10 uCi/ul P32 UTP (800Ci/mmol) (Dupont NEN - #NEG507X), or 1 ul for loading control probes
1ul RNA polymerase SP6,T7 or T3 (concentration varies by vendor)
Total Volume ~20ul.