Method to analyze transformation efficiency of competent E. coli

Transformation efficiency

The transformation efficiency is defined as the number of transformants generated per µg of supercoiled plasmid DNA used in the transformation reaction.

Transformation efficiency is calculated using the formula below-

=   Number of Colonies on Plate    X 1000 ng/µg

    Amount of DNA plated (ng)

Example 1 -

Number of Colonies on Plate- 105,

Amount of DNA plated- 0.1 ng

=    105   X 1000 ng/µg

     0.1

=   1.05 X 10⁶ cfu/µg

cfu- colony forming units

Example 2 -

Number of Colonies on Plate- 206,

Amount of DNA plated- 0.1 ng

=    206   X 1000 ng/µg

     0.1

=   2.06 X 10⁶ cfu/µg

cfu- colony forming units

Scientific Publishing- Becoming much easier or difficult?

Here are some links to know more about-

Article retractions

http://retractionwatch.com/

Potential, possible or probably predatory open-access publishers

http://scholarlyoa.com/publishers/

Primer Designing- Points to be considered

1. Primer length- while designing primers for normal PCR, primer length should be 18-22 bp.

2. Addition of Restriction Enzyme site with additional bases-

While calculating Tm, No need to consider additional bases or RE cutting site included in primer. RE sites should be added to 5’ end of forward primer and to 3’ end of reverse primer.

3. While calculating Tm value- Primers with Tm 52-58 ⁰C produces better results. Primers with melting temperatures above 65^oC have a tendency for secondary annealing.

The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. The difference of 5 ^oC or more can lead no amplification.

Nearest neighbor thermodynamic theory-

Formula for primer Tm calculation:

Melting Temperature Tm(^oK)={ΔH/ ΔS + R ln(C)}, Or Melting Temperature Tm(^oC) = {ΔH/ ΔS + R ln(C)} - 273.15

Where ΔH (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pairs enthalpy values of each nearest neighbor base pair.

ΔS (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up all the di-nucleotide pairs entropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all calculations.

ΔS (salt correction) = ΔS (1M NaCl )+ 0.368 x N x ln([Na+])

Where

N is the number of nucleotide pairs in the primer ( primer length -1).

[Na+] is salt equivalent in mM.

[Na+] calculation:

[Na+] = Monovalent ion concentration +4 x free Mg2+.

Generally Wallace Rule is used to roughly estimate Tm.

Tm = 4 (G + C) + 2(A + T)

4. GC content- GC content should be 50-60 % in designed primers.

The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.

Global Warming and its Impact on World Food Production. United Graduate School of Agricultural Sciences-EU (Kagawa, Kochi and Ehime university), Japan

Korea-Germany Joint Symposium on Plant Biotechnology

I am pleasure to share the experience that I had in last two days in symposium.

The research data presented by German scientists and Korean Scientist was really different.

Main differences in the research areas of two countries can be stated as follows:

1. Germans have considered very big pictures and goals with expected application oriented results. On the other hand very few Korean researchers could give such kind of data.

2. The strong point of Korean research is that they focus on small points and make a good story to be published in high impact factor journals.

3. As compare to Korea, Germans have more high throughput techniques and they have many collaborations across globe to carry out specialized tasks.

4. Germans are very strong in Genomics and utilizing sequencing technologies while Koreans are very sharp to extract available data form database for their research.

Some of the talks that impressed me are summarize below:

1. Seasonal flowering in annual and perennial plants. By George Coupland (MPIZ)

2. Towards a blueprint for C4 photosynthesis derived from comparative transcriptomics of closely related C3 and C4 species. By Andreas Weber (University of Dusseldorf)

3. Molecular, biochemical and cellular studies of E3 ubiquitin ligases in response to drought stress in Arabidopsis. By Woo Teak  Kim (YONSEI University)

4. Barley- at the transition to genomics based crop improvement? By Nils Stein (IPK Gaterslenben)

5. Iron fortification of rice seeds through activation of Nicotinamine Synthase gene. By Gynheung An (Kyunghee University)

BIOCHEMISTRY: A Never-Ending Story

Britt-Marie Sjöberg
Department of Molecular Biology and Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden

More than 50 years ago, Reichard and colleagues elucidated how cells make their DNA building blocks—the deoxyribonucleotides or dNTPs (1). They found that the enzyme ribonucleotide reductase (RNR) converts ribonucleotides (RNA building blocks) to corresponding dNTPs. One would expect that such a central pathway for all living cells would be meticulously mapped by now. Yes—and no. Researchers have described several classes and subclasses of RNRs (see the figure) that appear to have the same evolutionary origin (2–5), but involve different chemical cofactors, and so enable cells to construct dNTPs under different environmental conditions. Whenever the field seems settled, however, fascinating new aspects appear (1, 2). On page 1526 of this issue, Boal et al. (3) report crystal structures of RNR complexes from the bacterium Escherichia coli that, together with earlier studies, confirm and neatly illuminate yet another way cells can construct dNTPs, this time with the help of manganese (Mn).

Science 17 September 2010:
Vol. 329. no. 5998, pp. 1475 - 1476
http://www.sciencemag.org/cgi/content/full/329/5998/1475

ARS 2010-Some clues received from Manjiri

1. UPOV poject objective and year
2. Pre-emergence herbicides
3. tundu disease caused due to
4. cause of exanthema disease
5. globodera is a pest found on which south indian crop
6. ore used in gold mining
7. everything will wait bt not agriculture given by
8. pits are present in which cells?
9. relation between saponification value and saturated , unsaturated fats
10. metabolic rate index value
11. carbon discrimination of particular crop
12. approximate no. of genes present in human genome
13. in rice which gene has been transferred for salt tolerence
14. lyposome mediated transformation has been successfully done in
15. vector size( BAC, YAC)
16. NPTII derived from
17. segration of gene of interest and antibiotic resistance gene in next progeny
18. farmers produces which seeds for further cultivation? (breeder , foundation)
19. michaleis menten equation
20. competitive inhibition and non, competitive inhibition
21. what will happen if, long day plants are grown in short day conditions?
22. C3, C4 PLANTS EXAMPLE
23. bacterial chromosome is activated in which light?
24. which light is used in remote sensing?
25. pulses are deficient in
26. what will happen if, light is interrupted in long day plants
27. chemolithotrophs means
28. H1N1 is similar to which virus?
29. which act as a cementing agent in soil particles?
30. stage in which plant grows from small leaves to organs
31. nitrifying bacteria
32. dna replication takes place in ---- phase of cell cycle?
33. what is phytotron?
34. metagenomics is a study of
35. epoxides are more reactive than other ethers (yes / no)
36. difference between chl A and chl. b is in which tetrapyrole ring? ( I or II)
37. T% is given and we hav to calculate c% in DNA
38. symbiotic association between rhizobium and legumes given by scientist
39. father of soil mocribiology
40. theory of natural selection given by