Preparing and running Licor gels for SSRs
Preparation of the gel solution mix:
Electrophoresis separation of the SSR markers was done with a LI-COR 4300 DNA analyser.
We used a gel matrix of 7% polyacrylamide. A working stock of the gel solution mix was prepared in advance and stored at 4 degrees C. Composition of the gel solution mix (for 500 ml working stock):
70 ml Long Ranger (50% stock; Lonza cat. 50611)
210 gr Urea
25 ml 20x TBE buffer
Double distilled H2O up to 500 ml
Dissolving urea is an endothermic process, therefore it is recommended to use pre-warmed water. After the solution has become clear, it should be filter sterilized and kept at 4 degrees C. The Long Ranger was added after filtration.
Preparation of the plates:
We used 25-cm LI-COR plates with 0.25 mm spacers and 64 well sharktooth LI-COR combs.
The plates were thoroughly cleaned with isopropanol, and the gel was poured using a plastic squeeze bottle with flip-top lid. We used 20 ml of gel solution mix for one gel. Polymerization was induced with 150 microliter of 10% freshly dissolved APS (ammonium persulfate) and 15 microliter TEMED.
For the electrophoresis run we used 1 liter of 1x TBE buffer.
Prior to loading, PCR products were denatured with 30 microliter loading buffer composed of formamide (Sigma cat. 47670) stained with bromophenol blue and vortexed.
Before loading the samples, a prerun of 10 minutes was performed with the following settings:
After the prerun was completed, we loaded 0.5 microliter of the samples. The run was performed with the same settings as the prerun. The gels were loaded multiple times with PCR products labelled with IRD 700 or IRD 800 primers. PCR products with different dyes were simultaneously loaded; whereas PCR products with the same dye were loaded at intervals of 1 hour. Gels could be reloaded for 6 – 8 hours.
At the end of the run the images collected in *.tiff format were converted in *.jpg format with Adobe Photoshop and imported in Microsoft Powerpoint for editing.
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SSR general conditions