Hi, I'm JIAHUI (:
These 1 week and a half, I've been really busy!! With all new things that I need to learn, there's so much to blog about!
These 1 week and a half, I've been really busy!! With all new things that I need to learn, there's so much to blog about!
Nanodrop (ND-1000 V3.1.2)
NanoDrop is a low-volume spectrophotometer that is used to measure concentrations of cDNA, proteins and RNA. It does not require the use of curvettes. It is also capable of using just 1μl of sample to measure a wide range of concentration, from as low as 5ng/μl to as high as 3000 ng/μl. (1)
Method:
1. Wash the metal tip with ethanol. Wipe clean with Kim wipes by dabbing.
2. Wash the metal tip with RNase-free water. Wipe clean with Kim wipes by dabbing.
3. Choose program ND-1000 V3.1.2.
4. Click on nucleic acid
5. Sample type: other (for cDNA)
6. Constant: 33
7. Add 1.5μl of RNase-free water and blank it.
8. After each addition of sample, wipe clean with Kim wipes.
9. Add 1.5μl of sample.
10. Record 3 results.
a. 260/280 (RNA/DNA ratio)
b. 260/230 (RNA/protein ratio)
c. Concentration (ng/μl)
RNA/DNA ratio and RNA/protein ratio = ideally more than 2
Depending on your sample type, the constant changes.
cDNA: constant = 33
RNA: constant = 40
1. University of Texas (2009). NanoDrop. Retrieved on 26th June 2009, from: http://www.icmb.utexas.edu/core/DNA/NanoDrop/Nanodrop.htm
It's that simple! (:
Done by, Lim Jia Hui
0703605F
Hey! My lab has this equipment too, kool rite? Coz it is very convenient to measure DNA or RNA concentration =D
ReplyDeletebtw, what do u mean by 'RNA water'? Is it RNase-free water? lolz
oh yah, I'm just thinking of the ideal RNA/DNA ratio and RNA/protein ratio, which should be >2. Do u know the reason why?
Vo Thu Hong Anh [Jess]
0705364H
Hi Jess!
ReplyDeleteSorry for the misunderstanding, RNA water refers to RNase-free water. It is very important to use RNase-free water, especially when the samples are RNA or DNA. I'm sure the user wouldn't want the samples to be degraded by RNase or DNase. (I’ve edited the RNA water to RNase-free water instead).
2nd Question,
As you know, to measure absorbance of RNA - wavelength is at 260nm, to measure absorbance of DNA - wavelength is at 280nm, and to measure absorbance of protein - wavelength is at 230nm.
Let me give you an example, if my RNA sample contains contamination from DNA, my RNA/DNA ratio would be lesser than 2, (eg my RNA reading = 5 and DNA reading is 8, the RNA/DNA ratio would be 0.625). This would indicate contamination. The same goes for RNA/protein ratio.
However, if my RNA sample is not contaminated, take example RNA reading = 5, DNA = 2, the RNA/DNA ratio would be 2.5.
However it is not every time you will get the number 2. Sometimes I can get a ratio of ~1.8. As long as the ratio isn't below 1, the results should be acceptable.
Hope this clears up the doubt (:
Cheers,
JOEY(:
03rd July 2009
1623PM
wohoooooo! Thanks for the explanation, my doubt is totally cleared =D
ReplyDeleteVo Thu Hong Anh [Jess]
0705364H
hello joey.
ReplyDeletewhat is constant? is it a programme function or a solution conc. ? or a type of solution?
am quite confused. =S
Tng Wess Lee
0702570C
TG02
Grp 10
Hi Wess (:
ReplyDeleteThe constant is a programme function, and it's quite standard. If you are measuring cDNA concentration, the constant will be set at 33. If you are measuring RNA, you set the constant at 40.
Hope this clears up the doubt (:
Cheers,
Lim Jia Hui
0703605F
oh...
ReplyDeleteokok. thanks !
cheers ! =D
wess
Hello Joey,
ReplyDeleteWhy did you measure protein at absorbance 230nm? Because from websites, the proteins are measured at 280nm and 230nm will measure other things such as salts. So wont it be inaccurate?
Thanks
Zhang'e
TG02
0704086H
Hi Zhang'e.
ReplyDeleteFor this experiment I did not measure protein at absorbance at 230nm. If you are referring to the example that I gave to Jess, it's referring to the ratio.
for ratio 260/280, this refers to the ratio of sample absorbance at 260 and 280nm. this ratio is used to assess purity of DNA and RNA. in any case of ratio is lower than 1.8 or 2, this may indicate contamination of proteins, phenol or other contaminants.
for ratio 260/230, this refers to ratio of sample absorbance at 260 and 230nm.this is a secondary measure of nucleic acid purity.
because i'm measuring cDNA concentration, i just add the sample to the machine and the ratios and concentration will be generated.
if i'm measuring protein concentration, I would choose protein A280 module software instead of nucleic acid.
basically the ratio calculation is the same as what we learnt in Molecular Genetics (I don't remember what module), whereby we place the sample in curvette and measure at OD at 260, 280 and 230nm and we perform the calculation to determine if there is any contamination.
hope this answers your question!! if I made you more confused, do comment again!! (:
Cheers,
LIM JIA HUI (JOEY)
TG01 Group 02,
0703605F