Hello everyone,

I have issues when doing a mycoplasma test with PCR (a routine we set in our lab to test the presence of mycoplasmas in other's cell lines), using a 1% agarose gel.

When taking the photo of the gel with the program "SyberSafe" of ImageLab, there are several small black dots onto the gel, looking like black sand / dust... I can see the bands but the gel has so many flaws, i'd like to get rid of this problem. It happens either i make a normal gel or a minigel.

I do the following protocol:

- Mycoplasma mix preparation for 1 sample : 1.25µL Myco 5', 1.25µL Myco 3', 5µL Tampon, 0.5µL dNTPs, 0.5µL Phire Hot (enzyme, added lastly), 14µL H2O

- Add 22.5µL of the mix + 2.5µL sample in PCR eppendorfs, vortex + spin down

- Lauch the PCR program :

95°C 4mn,

(95°C 1mn, 57°C 45s, 78°C 1mn) * 30 cycles,

71°C 10mn,

4°C hold

- 1% agarose gel preparation : weight 0,50g of ultrapure agarose diluted into 50ml of TAE 1X using microwave (2mn at 360W)

- Cool down the bottle with tap water, transfer to an erlenmeyer and add 5µL of SyberSafe (crosslinking DNA agent), homogeneize gently before pouring into the tank with the comb. Let it polymerize for 30 minutes

- Add 5µL staining blue to each of the samples

- Load 10µL of sample into the wells

- Migrate for 30 minutes

- Reveal on a UV plate with the program "SyberSafe" of ImageLab

If anyone has suggestions about what causes the gel to have this "black sand" onto it instead of being smooth of the image ...

I precise a colleagues watched me (twice!) doing it to look for something i would be doing wrong, but found nothing and doesn't understand either.

Thanks in advance,

Gaëlle