2.A single membrane or a maximum of two is washed in a close able box with a volume of about 200mL for about 30 minutes.
3.Change buffer 4 or 5 times.
4.Once membrane has been washed, wrap in Saran wrap, make sure there are no wrinkles and expose to film.
II Hybridization (Old method, not recommended)Prehybridize filter in 2mL 5X "P" buffer
5mL Formamide deionized (optional)
1mL Salmon Sperm DNA 1mg/mL
0.58g NaCl (do not vortex, mix gently)
Dilute to 10mL with ddH
2O
1.Seal filter in Nylon bag without bubbles. Incubate for at least 30 minutes @65°C. (42°C with Formamide).
2.Add probe to bag, reseal and double bag. Incubate at least 6hrs at 65°C with slow shaking. NOTE: Probe concentration should not exceed 5-10ng/mL to avoid background.
Washing
1.Once hybridization is complete wash filter as follows.
2.Wash filter 2X with 250mL of wash A for 5 minutes at room temperature with gentle shaking.
3.Wash filter 2X with 250mL of Wash B for 30 minutes at 60°C.
4.Wash 2X with 250mL of 1:10 Wash A for 30 minutes at room temperature with gentle shaking.
5.Double wrap damp filter in plastic Wrap and expose to film.
End labeling of 8/HindIII
Digest 3μg DNA with 8/HindIII
Sample digest reaction
5μL stock DNA 1μL 10X Salt
1μL HindIII (10U/μL)
3μL ddH2O
Digest in the 37°C for 60 minutes
1.Add 20 μL TE pH 8.0
2.10μL A- Mix
3.1μL Klenow (5 units)
4.1μL 32P-dATP>3000
5.Spin and incubate @ RT for 30 minutes.
6.Add 110μL TE (150μL Final Volume)
7.Phenol extract
8.Run aqueous layer over spin column
9.Count 1μL
10.Store at -20°C properly shielded.
Probe Yip 56 with Random Primers
Random 6 Mer @ 1μg/μL concentration.We use Yip 56 @ 0.1μg/μL
1.2μL Yip 56
2.2.5μL 10X Klenow Salt
3.2.5μL Random 6 Mer
4.10μL ddH2O
5.Heat to 95°C for 5 minutes, plunge on ice.
6.Add 8μL A- Mix
7.Add 5 U Klenow
8.Add 2μL dATP 32P >3000
9.Incubate @ RT x 2 hrs.
10.Add 100μL TE
11.Pass over spin column, equilibrated with TE
12.Heat to 95°C for 5 minutes, plunge on ice.
13.Count 1μL .
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