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Alright! Thank you so much..

Hi CharonY, Thank you for the response. It helped me to understand the procedure more clearly! but I wonder what is the importance of letting the samples sit at RT for a while and then centrifuge??

Alriht..let me know if you get any further information regarding the issue, Thank you for the helpful responses

Thank you for the response but I still am confused as I didn't add water to the sample! Here is the procedure I followed, 1. Lysed and homoginized the tissue sample with 1 ml of qiazol 2. spun the sample through qia shredder tube, 1000 RPM, 1 min, @ RT 3. added 200 micro litters of chloroform 4. vortex well 5. spun at 9400 RPM, 15 min, @4'C And after the 5th step I am supposed to see 3 phases of RNA, DNA and Protein....And I did not see any RNA (Aqueous phase!)...So can you tell me where did I do anything wrong in this 5 step protocol?? Also I have another question regarding RNA quality analysis, I collected some RNA and quantified it using nanodrop, concentrations are good and 260/280 ratios are also good : between 1.8 and 2 But when I ran agarose gel(1%, with 5 micro liter of ethidium bromide, Samples: 1 micro gram of samples with 6X loading buffer ), all the samples gave smear.....no bands! Do you have any idea why did that happen??

Hello, I have phase separation problems after homogenization and chloroform addition. Some of the samples have a very thick interphase and little/no aqueous phase after centrifugation and once inverted aqueous phase. What will be the cause of this situation and how should I overcome this?