I would like to use hybridization between a probe and target DNA. We usually use PBS Buffer as a hybridization buffer. I want to try another solution. Any recommendations?
Hi!
In our lab we successfully use 20x SSC buffer with addtion of 50X Denhardt's solution (mix of denatured ssDNA and BSA). Denhardt solution blocks non-specific DNA hybridizations. You can also speed up your hybridizations with so called Hybridization Rate Accelerators which are dextran sulfate, ficoll, and polyethylene glycol. They can be used at concentration 10% or 5% in case of high level of non-specific hybridization.
Very detailed discussions on the conditions for DNA hybridization and the buffer components and their roles you can found here:
http://share.pdfonline.com/a7a88937568241fca5f05b28c0443571/southern%20hybridisation.htm
and here
http://www.opsdiagnostics.com/notes/ranpri/rpdnahyb.htm
Good luck!
Hi!
You can try "Church" buffer (GEORGE M. CHURCH* AND WALTER GILBERT*. Genomic sequencing. PNAS, Vol. 81, pp. 1991-1995, 1984).
Good luck!
Regards,
Evgeny.
Hi!
In our lab we successfully use 20x SSC buffer with addtion of 50X Denhardt's solution (mix of denatured ssDNA and BSA). Denhardt solution blocks non-specific DNA hybridizations. You can also speed up your hybridizations with so called Hybridization Rate Accelerators which are dextran sulfate, ficoll, and polyethylene glycol. They can be used at concentration 10% or 5% in case of high level of non-specific hybridization.
Very detailed discussions on the conditions for DNA hybridization and the buffer components and their roles you can found here:
http://share.pdfonline.com/a7a88937568241fca5f05b28c0443571/southern%20hybridisation.htm
and here
http://www.opsdiagnostics.com/notes/ranpri/rpdnahyb.htm
Good luck!
In biochemistry and molecular biology, the saline-sodium citrate (SSC) buffer is used as a hybridization buffer...
http://en.wikipedia.org/wiki/SSC_buffer
I agree... Church buffer is best!!! I use without BSA.
Good luck!
In our hands, we obtained the best result using PerfectHyb Plus (SIGMA)
Hello, you can use "ECL Direct Nucleic Acid Labeling and Direct Detecting (RPN 3000 Amersham)". We successfully used it. M. Santagati et al. 2012. We performed also a membrane prewash with SSC 5X and a post-hybridization wash with SSC 2X.
Good luck, Regards,
Francesco
Hello Seda.
I have used both commercial and lab-made buffers and find UltraHyb (Ambion/Life Technologies; #AM8670) to be the best by far. Not only is the level of background significantly reduced, but the need for in-depth optimisation is minimised. Essentially it is a fool-proof buffer system.
Good luck,
Wayne
Dear colleague, the choice of hybridisation buffer often depends on the type of membrane, hybridisation conditions (temperature) and even the size of probe. In our lab we are using a slighly modified CHurch-Gilbert buffer: 0.5M Na-phosphate, pH7.5 supplemented with 10 mM EDTA and 7% SDS that seems to be quite universal. The buffer works well with all charged and non-charged nylon membranes and at hybridisation temperatures ranging 42-80 C. Several kb-long DNA fragments as well as short as 25 bp oligos can be used as probes. With short probes the washing conditions should be modified,however. The buffer should be slightly heated before the use since SDS precipitates at room temperature upon the storage.
Hi, we use a Hybridization buffer containing SSc, SDS Laurylsarcosine and a blocking reagent from Roche.
The recipe is
Component Stock solution Vol Final concentration
SSC 20x (25 ml) 5x
Laurylsarcosine (10%) 1ml 0,1%
SDS (10%) 200 ul 0,02%
Blocking reagent (Roche) 1 gr
Final volume 100 ml
Good Luck
The Church&Gilbert hybe buffer (0.5-1 M Na/PO4 pH 6.8, 10 mM EDTA, 7% SDS) works very well in most conditions, provided it is warmed befoe use.
For Southern blot I recommend Church buffer, since SDS prevents non-specific binding of probe to the membrane.
Best regards
Church-based buffer, for pre-hyb and hybridazation. Foolproof.
My recipe:
Prepare
Na2HPO4 - 0.5M
NaH2PO4 - 0.5M
For 500mL
342 mL Na2HPO4 0.5M
158 mL NaH2PO4 0.5M
lead to pH 7.4
+50g SDS
+ 10 mL 0.5M EDTA pH 8.0
Good luck!
Hi Seda: I have obtained good results with the following buffer, also I have a whole protocol, which is working good with DNA and plasmid probe if you want to try too. All the best for your future work.
-Prepare the hybridization mixture as follows:
Deionized formamide 10 µl
20X SSC 2 µl
Sheared salmon sperm DNA (10 mg/ml) 2 µl
Probe DNA 1-2 µl
H2O ? µl
50% dextran sulfate 4 µl
Total 20 µl
Notes: Use H2O to adjust the total volume to 20 µl. Be sure that you are preparing the right amount of hybridization mixture instead of the number of slide (10 µl will be applied to each slide with a 18 ´ 18 mm cover slip). Make sure the solution is well mixed because the 50% dextran sulfate is very sticky.
---------------------------------------------
20X SSC Recipe
Following SSC buffer recipe is for total volume of 1 liter:
Component Amount 20X Stock Concentration Final 1X Concentration
NaCl 175.3 g 3 M 150 mM
Na3Citrate× 88.2 g 300 mM 15 mM
2H2O
-----------------------------------------------
Dissolve 175.3 g of NaCl and 88.2 g of Sodium Citrate in 800 ml water.
Adjust the pH to 7.0 with a few drops of 14 N solution of HCl.
Adjust the volume to 1 liter with ultrapure water.
Dispense into aliquots. Sterilize by autoclaving
We are using the following buffer
Prehybridization Mixture
20 x SSC 3.0 ml
100 x Denhardt 1.5 ml
Denatured salmon DNA (10mg/ml) 0.3 ml
Formamide 12.0 ml
Distilled water 13.2 ml
total 30.0 ml
Hybridization Mixture
prehybridization Mixture 10 ml
Dextran sulfate 0.25 g
total 10.0 ml
20×SSC:
3 M NaCl
0.3 mM 3Na-citraite•H2O
100 x Denhardt :
2% bovine serum alubmin (fraction V)
2% polyvinylpyrrolidone K-30
2% Ficoll 400
Does anyone have experience about hybridization buffer for fluorescent in situ hybridization (FISH) ? Why I have a lot of unspecific background with p53 probes? Many thanks!
There might several reasons for unacceptable background in FISH:
1. Poor probe quality - check labelling efficiency. For single copy gene the probe has to be well-labelled with high amount of incorporated fluorochrome.
2. Ceck if the hybridisation buffer contains heterologous DNA that helps to reduce probe non-specific binding.
I would go for 1M NaCl, TE1X plus 100 mM MgCl2 as we have already used it in our lab for DNA hybridization.
We are using a modified Church-Gilbert buffer: 0.25M sodium phosphate buffer pH 7.0 supplemented with 7% sodium dodecyl sulfate (SDS). The advantages are: no BSA or DNA additives are needed, works in wide range of temperatures (55-80 C), short (30 min) prehybridisation times, cost effectivness and is relatively simple to prepare. .
If your probe is LSI :
Hybridization buffer : 50% FA, 2 SSC, 10% dextran sulfate (pH 7)
Centromere or telomere probe: 55% FA, 2 SSC, 10% dextran sulfate (pH 7)
I just prepared the Church-Gilbert buffer as describe below:
http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8876.full?text_only=true
The colour turned out to be light yellow; is this the right colour?
Hamid, normally the Church-Gilbert buffer is bright, colourless.. The light yellow colour likely originates from serum albumin that sometimes contains contamination by blood components. In my opinion hybridisation is not influenced by these inpurities.
Ales Kovarik, Can the modified Church-Gilbert buffer [0.25M sodium phosphate buffer pH 7.0 supplemented with 7% sodium dodecyl sulfate (SDS)] be used as hybridization buffer for Fluorescence in situ hybridization (FISH) ?
which catalog number of sigma is better to use sodium chloride for 20XSSC?
Answer to Ruper and Yumlemban: No, this buffer is not suitbable fro FISH since it is used at elevated temperatures (55-70 C). Such a temperature would damage the chromosomes. Sorry for the late response but the message has been reported to me only recently.
Answer to Nino Oniashvili:
The ready-to-use 20x SSC concentrate from Sigma works fine. However it is expensive. We routinely mix 20xSSC from the components: Sodium citrate and NaCl. To prepare one liter of 20 x SSC one should mix:
-175 g NaCl
- 88 g sodium citrate dihydrate (Mw - 294)
Mix on a stirrer and adjust the pH with 2mL HCl
The cost is about one tenth of that of the premixed buffer...
https://www.sigmaaldrich.com/catalog/product/sigma/sre0068?lang=en®ion=CZ&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold3-2
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