anti-DIG magnetic particles
HilarioE at landcare.cri.nz
Thu Mar 30 04:13:16 EST 2000
I'm trying to clone a 3.1 kb restriction fragment using a DIG-labelled
probe and anti-DIG magnetic particles. But so far no luck. I know the
DIG-probe does bind to the anti-dig particles, since eluting with GuHCl
strips off a little bit of the probe. However, no restriction fragment
is enriched at all (checked by Southern blot analysis). I suspect the
problem is in the hybridization conditions.
At the end of this email you can find the protocol I designed.
Does anyone have any advice for solving this problem?
I really appreciate all your help.
Regards from New Zealand,
<hilarioe at landcare.cri.nz>
Set up two tubes as follows:
2 ul (~40 ng) DIG p10 probe in HEN 7.5 buffer (Hepes 50 mM, EDTA 1 mM,
NaCl 0.5 M, pH 7.5)
4 ul (~5 ug) P. agarici gDNA EcoRI digested, in HEN 7.5
4 ul HEN 7.5 buffer
1. Boil 5 min, ice cool for 5 min. Spin down and transfer to 0.2 ml
2. Hybridize at 65°C for >17h
1. Wash 2 sets of 50 ul of antiDIG-magnetic particles three times with
TEN100 (Tris 10 mM, EDTA 1 mM, NaCl 0.1M, pH 7.5) 100 ul, each time
2. Do the last resuspension in 40 ul TE buffer pH 8.0
3. Add the 10 ul of hybridization mix to the 40 ul of washed particles
(NaCl final concentration is 0.1 M). Incubate 30 min at room
temperature. Gently tap the tube to keep particles resuspended.
4. Collect supernatant and save
5. Wash two times with 50 ul TEN1000 (Tris 10 mM, EDTA 1 mM, NaCl 1 M,
pH 7.5). Collect both washes in the same tube
6. Elute option 1) with 50 ul TE buffer pH 8.0 at 80°C, 5 min
option 2) with 50 ul Guandidine Hydrochloride 6 M at room
temperature, 5 min
Note: the particles eluted with GuHCl look damaged. The particles
eluted with TE at 80°C look normal.
7.Precipitate all samples with 1 ul 10mg/ml tRNA, 1/10 vol 3M NaAc pH
5.2, 2 vols ETOH, -20°C, overnight.
8. Wash and resuspend in 20 ul TE pH 8.0 and load all. Do a Southern
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