I extracted RNA, converted it into cDNA. Then, I used Qiagen HotStarTaq Plus Master Mix Kit for PCR. Then I run AGE at 80 Volt, 67 mA in 45 minutes (constant: V). I want to check whether my 4 primers are working or not, so first lane ladder, second till fifth lane samples plus forward reverse primers, and another four lanes of NTC. I did not add loading dye to dna ladder, but I used 1 ul loading dye and resuspend in 5 ul of sample/NTC.
I could see the bands without uv (still none for dna ladder) but with uv there's none.
This is what it looks like without UV. I used 50ml TBE with 0.75g agarose powder to prepare the gel. Added etbr free stain into the gel and cool it down. I used TBE buffer to run the gel. For the cDNA pcr, I followed the Qiagen protocol but I omit adding Coralload concentrate (used it before but didn't work, no band at all), and total up each pcr tubes has a reaction volume of 10ul.
Try letting it soak in the staining solution in buffer for a few hours. You obviously had good separation of your loading dye. Once the ladder looks bright, see if you have any other bands.
@paulrutland I used Yeastern 1uL of Etb 'out' nucleic acid staining for 50ml agarose in 1% TBE.
@katieaburnette Thank you. Does that mean I need to load the ladder and sample, and let them soak in the 1% TBE buffer? I told my postgrad about the problem, and she said the DNA ladder I used (Promega 100bp ladder) did not have any dye (it was colorless). That might be the problem also. Will try again and update thw results. Thank you!
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