I need to make some loading buffer for my gels, and I have never made any before. We use bromophenol blue, and make a 6x buffer concentration. I am just doing simple 1% agar gels with ethidium bromide to look for DNA. If you have a simple recipe you could share, it would be greatly appreciated.
Thanks!
0.2 g Bromophenol Blue
6 ml 50% glycerol
4 ml milipore water
I like using Orange G (runs ~50 bp in 1% gels). I make a 10X solution which is simply:
100 mg Orange G
15 ml glycerol
~35 ml water (final volume to 50 ml)
10 X glycerol dye
Bromophenol Blue 200 mg
Xylene Cyanol FF 50 mg
0.5M EDTA (~pH 8) 20 ml
glycerol 90 ml
water---up to 120 ml (add about 10 ml)
mix well, autoclave
I ended up using something like:
0.25 g Bromophenol Blue
6 ml 50% glycerol
4 ml milipore water
NaOH to adjust pH
@Masoud
Components like sucrose, glycerol or Ficoll that are found in different gel loading formulations all have the same role--to make the sample more dense than water so it will sink to the bottom of the gel well as you load.
25 mg Bromophenol Blue, 25 mg Xylene cyanol FF, 4 gram of sucrose in 7 ml of water. Mix well and adjust the total volume to 10 ml using milliQ water
[quote]I want to know if autoclaving is important for loading dye or no[/quote]
Answer: Not really. All components added to the loading dye are easily soluble in water after all. And any impurities still left undissolved can easily be removed using a 0.22 um filter which gives you an equally sterile batch much quicker for further storage.
1. Weighed 121 g of Tris-HCl and put into a 1 liter flask and add Aquabida(H2O) up to 1L and dissolved it by stirring
2. Made dilution (1:10) of 1L solution of Tris-HCl into another flask
3. Measured 60 ml of 100% Glycerin into another flask
4. Add 3 ml of 3% Bromophenol Blue into 60 ml of Glycerin
5. Add 10 ml of 1:10 dilution of 1L Tris-HCl to the step 4 and stirring properly and label it and used as 6X loading dye
What's the rule of EDTA in loading buffer? Some recipes are using EDTA
Some suppliers say that the EDTA is included in the solution to protect sample from nuclease degradation. But I still wonder whether its still necessary in such a short time of gel loading.
I found a recipe with only
Bromphenoleblue (0.25%)
Glycerol (30%)
What is the advantage of adding Xylene cyanol FF or Sucrose?
The Xylene Cyanol FF and Bromophenol Blue are used for monitoring the agarose gel while it is running, so you can use just one of them to predict your desired DNA migration@Ivo Röwekamp
This sounds the easiest and most convenient. I have been using this recipe for last 20 years and did not go wrong.
So basically only 30% of glycerol is needed to load the samples, right?
Janine Liedtke
If, for some reason, you do not want to add any dye to your sample when you load it, then yes, all you need to add is something that will make it sink in the well (and not just float away). This can be achieved with 30% glycerol, 40% sucrose, or 25% Ficoll. In all cases, you would add 1/5 to 1/10 volume of these to your sample.
Nawroz Abdulrazzak is wrong: it gives 10x more brightness that covers the 700bp DNA and DNA is not visible under dye. it should be .02 g for 10ml which is 0.2%.
Elizabeth E Traylor How much NaOH did you add? and did it work for you?
- Badges
- Science method