Skip to main content

Cot Value Analysis

Hi,
Wish you a good time :) Molecular biology! I was taught about "cot value analysis" in my molecular biology class. Here, you could read something, which I know about cot value.

Cot (C not (zero) t) value analysis:
This analysis uses the DNA denaturation and renaturation kinetics. This is used for measuring the repetitive DNA sequence in a given DNA sample. This analysis has application like separating repetitive sequences from mixture of DNA sample. Okay, let us discuss the procedure now.

Note:
Cot value = (C zero) * (Time) * (constant representing the effect of cation in the buffer on renaturation)
where,
c zero -> DNA concentration (mol/l)
T - Renaturation time (sec)

Procedure:
  1. Denature the DNA sample by heating
  2. Cool and renature the sample, note renaturing extent (as cot value) at particular temperatures until a particular cot value is reached.
  3. Now, dilute this sample with sodium phosphate buffer- SPB (0.03M - at this buffer concentration the DNA bind to hydroxyl apatite properly)
  4. Bind this buffer solution with DNA to hydroxylapatite(HAP) column.
  5. 0.12M SPB is now used for eluting Single stranded DNA from HAP; Collect the Single stranded DNA.
  6. 0.50M SPB is then used for eluting double stranded DNA which is also collected.
  7. Volumes of Single and double stranded DNA collected is measured.
  8. 0.9ml of the centrifuged single stranded DNA is mixed with 0.1 ml of  10N KOH. This addition of KOH denatures the double stranded DNA (if present). Do this to the double stranded DNA sample also.
  9. Absorption values A260 (adjusted for light scatter at 360nm) are obtained for both the samples prepared (mentioned in the previous point)
Percentage of single stranded DNA could be calculated with the help of the following formula:

[(Vss x Ass) x 100] ÷ [(Vss x Ass) + (Vds x Ads)] = % ssDNA
where,
Vss = total volume of single-strand fraction, 
Vds = total volume of double-strand fraction, 
Ass = A260(adjusted for light scatter) for the KOH-denatured single-strand fraction,
Ads = A260 (adjusted for light scatter) for the KOH denatured double-strand fraction.

Background:
  • The rate of renaturation of repetitive sequences will be high comparing the rate of renaturation of non repetitive or less repetitive sequences.
  • This is because, the possibility of a repetitive sequence to find it's complementary strand is greater than that of a non repetitive strand. (Hope you got it)
For detailed explanation refer "here"

Got any doubts? Kindly comment I'll try to answer.

Bye, Take CARE! :) 



Comments

  1. Cool and renature the sample, note renaturing extent (as cot value) at particular temperatures until a particular cot value is reached.
    How is this step done?

    ReplyDelete
    Replies
    1. Cooling can be done by using a water bath, simple? And using the formula for cot value calculation, get the cot values. For detailed explanation refer the following link:

      http://www.mgel.msstate.edu/pdf/cot.pdf

      Delete

Post a Comment

Popular posts from this blog

Lowry Assay Principle and procedure

Though there are several protein assays available, the most preferred one in many laboratories is "Lowry assay". It is effective in the concentration range of 0.01 mg/ml to 1 mg/ml. And, as an additional info, the paper published describing the procedure and principle of Lowry Assay is the most cited paper in the scientific history. (Feeling like, "Wow! I want to publish one to compete with Oliver.H. Lowry"???  :P) Why Lowry?   Though there are several other protein assays, mostly Lowry assay is used in many laboratories. The reasons for preferring Lowry are: sensitivity of the assay, highly reproducible, cost effective, easy to perform. Biuret assay is generally used for higher protein concentrations like tissue samples but, Lowry for less concentrated samples and hence used in most of the molecular biology laboratories where there will be need for assaying comparatively less concentrated protein samples (in most cases where we attempt to produce enzymes). Oth...

Sea Butterfly!

Neih hou!  Don't roll you eyes wondering what it is! "Neih hou" is how you say "hello" in Cantonese. Guess what, I am in Hong Kong and therefore the language Cantonese. I came to Hong Kong this summer as an intern before officially joining as a PhD Student. This is my second experience abroad, far away from home, new language, new culture, I expected me to have a "really" bad cultural shock, but, actually I experienced only 50% of what I expected. For a girl who have used the marina beach only for eating "sundal", bikini in beach was a shock (FEEL FREE TO JUDGE ME!). The first time, I went to the big wave beach here, I was the only one who was totally covered, when everyone was enjoying their Beer, I was slowly sipping my lemon juice (THE ODD ONE OUT!). I realized how beautifully different the world outside is! There is no single standard for right or wrong, it varies in different countries, in different regions around the world. And it ...

Fire in the lab!!! BE CAREFUL!!!! A Lesson learnt!

   That day, we were working in our lab under laminar air flow chamber. we were streaking our plates with E.coli. For sterilizing the loop, we were using 70% ethanol. We also sterilized the L rod which we used for spreading , using the same Ethanol.  One by one, we started streaking and spreading. I completed my turn, and sterilized the L rod by dipping in ethanol once and shown in flame, and placed it in a tray.Mam instructed, " Ethanol will catch fire , if you place the hot rod over it" . One of my friend, did spreading after my turn, she too dipped the rod in ethanol, flamed the rod and instead of placing in the tray she misplaced the hot rod in the ethanol plate!!!! " oooo... fire fire..." immediately ethanol caught fire..!!! My friend who misplaced., she started crying... Mam shouted, " hey girls, guys run out of the lab...," as the tube which carries LPG for the flame was very close to fire. Suddenly, our lab technician acte...