我想从革兰氏阳性菌中分离RNA。你们推荐使用什么产品?是否需要改进裂解方案?
对于细菌样品,您可使用我们的TRIzol Max细菌RNA分离试剂盒(货号16096040),该试剂盒在匀浆时含有额外的试剂,有助于细菌细胞壁的裂解。您也可以使用我们的PureLinkRNA小量提取试剂盒(货号12183018A),使用该试剂盒时,需要准备溶菌酶溶液以溶解细胞壁。
I'd like to isolate RNA from gram-positive bacteria. What products can you recommend for me? Is a modified lysis protocol needed?
For bacterial samples, you could use our TRIzol Max Bacterial RNA Isolation Kit (Cat. No. 16096040), that contains an extra reagent during homogenization to help with lysis of the bacterial cell walls. You could also use our PureLink RNA Mini Kit (Cat. No. 12183018A), for which you would need to prepare a lyosyme solution to lyse the cell wall.
The TRIzol Reagent protocol specifies centrifugation speeds of 12,000 x g for 10 mins for RNA precipitation, but my centrifuge only goes up to 5,000 x g. Can I still perform my experiment?
Yes, centrifugation speeds as low as 5,000 to 6,000 x g have been used, but the centrifugation time should be doubled to get the expected yields.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
I accidentally added too much chloroform to my TRIzol Reagent reaction. What should I do?
If a large amount of chloroform was inadvertently added, you can add more TRIzol Reagent so that the ratio of 0.2 mL chloroform:1 mL TRIzol Reagent is maintained. If too much chloroform is added, this can drive the DNA, and eventually the protein, into the aqueous phase.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.
I inadvertently added isopropanol instead of chloroform. What should I do?
If isopropanol is inadvertently added at this step instead of chloroform, add more isopropanol to precipitate everything, then resuspend the pellet in TRIzol Reagent and use the protocol as specified. RNA yields will be compromised, but it may be possible to obtain a product in RT-PCR. A detailed protocol follows:
(1) Add more isopropanol so that the total volume of isopropanol equals the volume of TRIzol Reagent used. Spin at 7500 x g for 10 min at 4 degrees C.
(2) Pour off supernatant; allow relatively compacted pellet to air dry (doesn't have to be completely dry, just reduce the volume of ispropanol).
(3) Estimate the size of the pellet in microliters; add at least 15–20 volumes of TRIzol Reagent (e.g., for a 100 µL pellet, add at least 1.5 mL TRIzol Reagent).
(4) Break the pellet up well (you may have to use a hand-held homogenizer). Store the solution for 10–15 min. at room temperature; every 5 min or so, shake it by hand to make certain it is well dispersed.
(5) Proceed with the TRIzol Reagent protocol as written (i.e., add chloroform). Results will not be optimal, but it may be possible to get a product in RT-PCR.
Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.