RNA isolation problem - (Sep/22/2008 )
QUOTE (Sarwat @ Oct 23 2008, 09:46 AM)
Hi,
I have a similar problem but the difference is that I saw a white pellet when I washed my cells with 75% Etoc (made in DEPC water). I air dried it for 20mins and the white pellet went invisible (see through in colour). Anothertube I was working on has teh white coloured pellet after air drying it for the same amount f time. To play safe, I dissolved both pellets in DEPC treated water and incuated in 60 deg water bath. I have not checked the concentration of both the RNA's but any idea why one pellet went from white to clear? Did I over dry my pellet?
I have a similar problem but the difference is that I saw a white pellet when I washed my cells with 75% Etoc (made in DEPC water). I air dried it for 20mins and the white pellet went invisible (see through in colour). Anothertube I was working on has teh white coloured pellet after air drying it for the same amount f time. To play safe, I dissolved both pellets in DEPC treated water and incuated in 60 deg water bath. I have not checked the concentration of both the RNA's but any idea why one pellet went from white to clear? Did I over dry my pellet?
Pelets turn to transparent, it's normal. But you should probably dry it less time. Most likely the white pelet had some stuff inside, that kept it white.
-Trof-
RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA.[1] Several methods are used in molecular biology to isolate RNA from samples, the most common of these is Guanidinium thiocyanate-phenol-chloroform extraction.[2][3] This method often uses a proprietary formulation of this reagent called Trizol.
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Sam
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-sam2008-
QUOTE (SYDNEY @ Oct 22 2008, 02:01 PM)
QUOTE (medstudent1 @ Oct 21 2008, 09:07 PM)
QUOTE (Trof @ Oct 20 2008, 08:51 AM)
medstudent1:
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
hi trof and all,
thanks for all of the advice. good pointer about putting the tube in the same direction each time. as an update, ive been using RNA-STAT 60 and things have been working decently. ive been able to extract rna (approximately 1 ug/ml) and subsequently make cDNA for qPCR. i am isolating rna from 6 well plates so you are right about the pellet - i have been going on "good faith" when pouring off my supernatant because as you mention i dont see a pellet.
Hi All,
Yes i use ice cold ethanol but 70% with DEPC. i do put the tubes in the same direction so i know which side most probably would because you know
literally the pellet is invisible. I want to ask when i drain teh liquid i usually do that with pipett , means i remove the supernatant with pipett, i sthat ok or just i should simply drained it from eppendorf?
sydney
I wonder if it is better to use ice cold chloroform and isopropanol as well?
-AS mikkel-
QUOTE (medstudent1 @ Oct 21 2008, 08:07 PM)
QUOTE (Trof @ Oct 20 2008, 08:51 AM)
medstudent1:
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
My comments:
- You didn't write if you actually get any RNA, but I'll assume you don't.
- We isolate from Trizol, but the procotol seems the same, rubyeye is right, definitelly use ice-cold ethanol (that's usually stated in the protocols) and important, dilute the ethanol (75%) in DEPC water only.
- For smaller amounts of sample (like a 6-well I would say) you usualy don't see the pelet, it's very small. In this case, put the tubes to centrifuge always in the same direction, so you know on which side the pelet would be (even invisible one), don't scratch that side with a pipette tip. Usually on a table centrifuge the pelet would be near the bottom (but not in the exact middle of it) on the distant edge from the center of the rotor.
- For the amount of cells you use and the amount of RNA-STAT 60 used, the final volume to resuspent in seems too much for me. We use for blood cell isolation 1ml of Trizol for cells, and then we disolve the final pelet in 10-15 ul od DEPC. Then we use about 1-2 ul for 20ul cDNA reaction.
hi trof and all,
thanks for all of the advice. good pointer about putting the tube in the same direction each time. as an update, ive been using RNA-STAT 60 and things have been working decently. ive been able to extract rna (approximately 1 ug/ml) and subsequently make cDNA for qPCR. i am isolating rna from 6 well plates so you are right about the pellet - i have been going on "good faith" when pouring off my supernatant because as you mention i dont see a pellet.
Hi all,
i'd advice u to try with blu glycogen in case of low amounts of RNA/cells. even if you know where the pellet is without seeing it u could lose it while washing. "isopropanol-step" pellets are difficoult to detach from the wall of the tube but when u wash with etOH the pellet is loosely attached and if u don't see it u could aspirete it while removing the liquid. just take care.
fizban
-Fizban-
QUOTE (Fizban @ Oct 24 2008, 05:03 PM)
Hi all,
i'd advice u to try with blu glycogen in case of low amounts of RNA/cells. even if you know where the pellet is without seeing it u could lose it while washing. "isopropanol-step" pellets are difficoult to detach from the wall of the tube but when u wash with etOH the pellet is loosely attached and if u don't see it u could aspirete it while removing the liquid. just take care.
fizban
i'd advice u to try with blu glycogen in case of low amounts of RNA/cells. even if you know where the pellet is without seeing it u could lose it while washing. "isopropanol-step" pellets are difficoult to detach from the wall of the tube but when u wash with etOH the pellet is loosely attached and if u don't see it u could aspirete it while removing the liquid. just take care.
fizban
oh.. good try... last time when i got low amount RNA, i also intend to try with glycogen... jus wonder, how much is the ratio when need to add in? what else need to add when add in glycogen?
thanx..
-parami-