I'm extracting bacterial DNA from human fecal samples. Planning to run my samples 1% agarose 0.5X TAE. I have lots of concerns so please bear with me.
1. The AGE apparatus is this one, https://www.capitolscientific.com/Thermo-Scientific-Owl-B2-BP-EasyCast-Mini-Gel-Horizontal-Electrophoresis-System-with-Buffer-Excha. There's no small trays. Just one big big tray with two slots for both combs. Anyone experienced working with this and any know how to making smaller gels? I'm planning to use heat-resistant plastic to mold smaller gels. Will that work?
2. I have very limited reagents, 100mL of 50X TAE, 20uL ladder, 15uL midori stain, lots of agarose and loading dye. So far I have only extracted from 10 samples (50uL per sample). I haven't optimized my extraction yet too due to the limited reagents for AGE. I also have yet to check if the ladder is still intact because it was delivered to me by our lab without an ice pack. Should I check the integrity of the ladder first and run with fewer samples (assuming I can make a 20mL small gel)? Or run all 10 samples in a 40mL big gel?
3. Regarding the limitation of my reagents, in our lab (not the lab I'm working at now where they have no experience on molbio) we recycle the stain by making a 20mL gel stain bath where lots of gels can be stained. I think it was 5uL for 20mL, I will confirm. Other labs directly put the stain on the gel solution, I dont remember right but was it after microwaving the agarose solution or before? Also of what I know it was 2uL per 40mL gel but I heard others do it by 0.5uL only. How do you save your gel stain in your labs?
You can find some suggestions interesting here:
1. You may use less than 1% Agarose gel (0.6-0.8%) for bacterial genomic DNA but 1X TAE is recommended.
2. I have never used other heat resistant plastic, because small glass tray was also available in my lab. But it is a good idea, so go ahead. Make sure that there won't be any leakage from the tray and arrange the comb inside the gel properly. Must be some gap from the bottom surface. All the best.
3. I think the DNA ladder will work because DNA solution is more stable and heat resistant than protein samples. You can run the DNA ladder and samples together. As you have limited source of DNA ladder, you can make it diluted. You can start with small gel volume with least number of DNA samples (suppose one DNA ladder and one or two genomic DNA samples). Once it will be standardized, you can run more number of samples.
4. I have always used pre-staining procedure with Ethidium bromide (EtBr) dye, but as you are used to post-staining, you can follow the same procedure. If you want to try pre-staining, I must suggest that nucleic acid staining dye is added after microwaving the agarose solution. At the first, allow to boil the agarose solution until it looks transparent homogenous solution, keep it to the room temperature until you can touch the bottom of the conical (as suggested by Oliver Krohn
), then add midori dye. As most of the nucleic acid dyes are heat and light sensitive, so you have to add this at lower temperature (not enough to get solidified), make the solution homogeneous again and then pour it down into the tray. According to the manufacture's protocol, 4-6 μl are recommended for the staining of 100 ml agarose gel, so you can use 0.8-1 μl midori stain for your electrophoresis. Also, I don't think that reusing gel is a good idea, you may use one gel two times, but in the next time, you will get poor resolution.I wish you all the best for your experiments.
Best regards. Princess Alyssa Abid
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