Multimerization Troubleshooting: No Luck in 2-3 Weeks

In summary, the conversation discusses the difficulties and potential solutions for creating a multimer from an oligo. The individual has tried various techniques such as annealing, phosphorylation, and ligation, but has only been able to produce a dimer. Suggestions are made to increase the concentration of DNA and use longer oligos to prevent circular products. The conversation ends with a request for an established protocol for this type of problem.
  • #1
karthik3k
149
0
Multimers :((

I have been trying this thing for past 2-3 weeks with no luck :((

:cry:

I am trying to make a multimer from an oligo.
This is how i did:
- Annealed the oligos
- Phosphorylated the sample (5' ends)
- ligated o/n

when i ran it on agarose it shows only a dimer. what can be the reason ?
any ideas ?

Should i use some different protocol ??
 
Biology news on Phys.org
  • #2
When I did ligations, I could see a ladder. My fragment was in a vector, which I excised with restriction enzymes. The result is sticky ends which ligate much better than blunt ends.

So either your ligation reaction is not efficient, or you are making circular DNA molecules. The get more linear product, you should ensure that the concentration of DNA in your ligation reaction is quite high. That way the DNA is more likely to come across a new DNA molecule to ligate to, than it is to its own free end. Another problem I see is that you use very small oligos. Mine were 100 bp, so the ends are further apart, thus less circular products.
 
  • #3
k.

do u have any established protocol for this kinda problem ? :confused:
 

1. What is multimerization troubleshooting and why is it important?

Multimerization troubleshooting is the process of identifying and resolving issues that prevent the successful formation of multimers, which are complexes of multiple molecules joined together. This is important because multimers play crucial roles in many biological processes and are often targets for drug development.

2. How long should multimerization troubleshooting take?

There is no set timeline for multimerization troubleshooting as it can vary depending on the specific system and issue at hand. However, if no progress is made after 2-3 weeks, it may be necessary to re-evaluate the experimental design or seek assistance from other researchers or experts.

3. What are some common causes of unsuccessful multimerization?

There can be many reasons for unsuccessful multimerization, including incorrect protein concentrations, incompatible buffer conditions, or improper mixing techniques. Other factors such as protein stability, post-translational modifications, and protein-protein interactions can also impact multimerization.

4. What steps can be taken to troubleshoot multimerization issues?

Some steps that can be taken to troubleshoot multimerization issues include adjusting protein concentrations, optimizing buffer conditions, changing mixing techniques, or using different purification methods. It may also be helpful to consult the literature or seek advice from experienced researchers.

5. How can multimerization be confirmed?

Multimerization can be confirmed using various techniques such as size exclusion chromatography, analytical ultracentrifugation, or native gel electrophoresis. Additionally, techniques such as co-immunoprecipitation or crosslinking assays can be used to detect protein-protein interactions, which are indicative of multimer formation.

Similar threads

Why is Ligation of Oligo A and Complement B Only Producing Monomers and Dimers?
    • Biology and Medical
Replies
17
Views
3K
A Number of unequal integers with sum S
    • Set Theory, Logic, Probability, Statistics
Replies
12
Views
966
Samsung double door fridge overload relay changed 3 times in 2 weeks
    • Electrical Engineering
Replies
12
Views
2K
MHB Tikz for f(x)=5x^2-2x and tangent line at 1,3
    • MATLAB, Maple, Mathematica, LaTeX
Replies
5
Views
2K
B Validating Darts Tournament Skill Levels: A Non-Intrusive Approach
    • General Math
Replies
4
Views
666
MHB Second week precal, x^1/2 + 3x^−1/2 = 54x^−3/2
    • General Math
Replies
6
Views
2K
Possible values an expression can take : ##\dfrac{x^2-x-6}{x-3}##
    • Precalculus Mathematics Homework Help
Replies
7
Views
759
I Runge-Kutta 4 w/ some sugar on the top: How to do error approximation?
    • Differential Equations
2
Replies
40
Views
537
MHB What is the Graph of $f(x)=3\cos(\pi x-2)+5$ with Amplitude and Period?
    • MATLAB, Maple, Mathematica, LaTeX
Replies
6
Views
1K
Complexity of this heap algorithm
    • Programming and Computer Science
Replies
1
Views
1K

Hot Threads

Recent Insights

Back
Top