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ravibiot's Content

There have been 25 items by ravibiot (Search limited from 18-February 19)

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#9709 PCR - No bands anymore in well functioning PCR

Posted by ravibiot on 02 January 2005 - 10:53 PM in Molecular Biology


do you check whether heating of buffer in electrophoresis, if so change the buffers regularly and try one more time.
Is your EtBr is working properly, are exposed much time to light


#9076 thermal cyclers

Posted by ravibiot on 01 December 2004 - 08:05 PM in Molecular Biology

yeah, this is the bad part of the thermocyclers.
even i have observed the same, i followed the thermal programme of perkin elmer (which has been published) i didn't get the amplification in eppendorf. finally i have standardize the thermal program (annealing temp.)got the the amplification. which i have checked by restriction analysis for the conformation.



#9059 A Digestion problem

Posted by ravibiot on 01 December 2004 - 09:15 AM in Molecular Biology

Yes it is !
it s necessary to check the conc. of insert and vector, and what you heard is right to take in 1:3 ratio (vestor : Insert). Pls. refer Molecular cloning by Maniatis, 1982 for detailed protocols.

Yes, as mentioned earlier by Janina, while purification try to elute in water. Coz... its assumed that EDTA some time interfere with digestion process.

Good luck


#9058 bubbles in LB plates

Posted by ravibiot on 01 December 2004 - 09:01 AM in Microbiology


In my experience, we can see lots of air bubbles in LB plates only when stored at 4 C for long periods, and brought to 37 C incubator.
To avoid bublles we can incubate upright for 1-2 h at 37 C and can be inverted.

In my consideration freshly prepared plates should not bubble up at any time. If its so, please do melt the LB agar in microwave not more than 2 min time. over heating of agar also will reault in bubble formation.

also, there is another reason like when you have LB agar in a conical flask which has been melted earlier to prepare plates and the left out LB agar when you reheat you will get bubbles.

Best wishes,

#9056 Anyone try Petrifilm?

Posted by ravibiot on 01 December 2004 - 08:38 AM in Microbiology


Its looks nice, but has to be tried once...


#9026 ligation and transformation problems! Urgent!

Posted by ravibiot on 30 November 2004 - 08:22 PM in Molecular Biology


May be you can check for linear ligation of PCR digest (since, your PCR product size is 400 bp and vector is 4 kb. So, the expected ligation product should be at 4.4 kb)
I suspect the problem lies in ligation. I used to do the ligation reaction at 4 C for 14-16 h (undisturbed), and i get efficient ligation. For transformation i take only 2 ul ligation product. I hope you will get good transformants.

Best regards,

#9025 peptide linkers

Posted by ravibiot on 30 November 2004 - 07:57 PM in Molecular Biology

Hi Tau,

His linkers can be added to the sequence of your interest in a simple way, There are commercial vectors available where the palsmid it self will have the His6 sequences. you can just insert your sequence in such a way that His-tag sequences come either at c-terminus or n-terminus site of your target sequence.
A minimum of 6 Histidine residues are needed for the affinity purification of you His- tagged protein on Ni+ column.

his6-tag vectors i used are pQE 60 and pET 28A+

Best regards,

#9014 Small competent cells

Posted by ravibiot on 30 November 2004 - 07:24 AM in Molecular Biology

well, i too had the same problem that you are facing now...
i substituted CaCl2 based competent cell preparation method (Gene cloning Maniatis, 1982) instead of RbCl2 based method and observed good results.
i hope that you too can try with the same. Also, you can check the LB medium age (use fresh plates), use appropriate final concentrations of selection pressure (antibiotic) and check the incubation temparature.

Best regards,

#9013 Cellulase plate assay

Posted by ravibiot on 30 November 2004 - 07:07 AM in Microbiology

U Are Welcome

#9012 Where are you from?

Posted by ravibiot on 30 November 2004 - 06:41 AM in Chit Chat



nice to meet all

#8965 Pepsi, Coca-cola successful in killng pests in INDIA

Posted by ravibiot on 28 November 2004 - 05:28 AM in Chit Chat

How far its safe to drink Pepsi and Coca-cola. Pesticide residues of Pepsi, and coca-cola were benn investigated by researhes of CFTRI, Mysore.
field trails of these soft-drinks on crop plants showed effective in killing the pests of cotton, chili and tobacco in india (Guntur districk).

for details ref. New indian express (28-11-2004)

So, take care


#8964 ligation and transformation

Posted by ravibiot on 28 November 2004 - 05:03 AM in Molecular Biology

i hope the antibiotic final conc. used should be increased to 60 ug/ml.
may be you can check the transformation again (6 kb).
Do you check for the plasmid by rapid lysis for the so called non-transformants (lawn).
you didn't mension about the +ve control. if you have not used you can also keep an appropriate positive control to check the success of you transformation experiment.

Best regards,

#8961 restriction digestion

Posted by ravibiot on 28 November 2004 - 04:29 AM in Molecular Biology

Check you have dissolved the DNA in water instead of TE buffer where EDTA interfere with restriction digestion.
Also prolong the incubation time

Hope you are taking sufficient enzyme for the digestion, pls check it alos

Best regards

#8960 Gas chromatography

Posted by ravibiot on 28 November 2004 - 03:59 AM in Microbiology

i do investigation in microbial ecology of hydrocarbon degrading bacteria.

All isolates i have degrading hydrocarbon are strictly aerobic. i wish to study the degradation kinetics (successive deplition of hydrocarbons in culture medium) of these bacterial strains. But, my doubt is hydrocarbons are volatile in nature, and when i start do the experiment i suspect the non-specific (colatilization) of hydrocarbons will mislead my results. and my bacteria are strictly aerobic (so, i cant plug with teflon lined stopper for culture flask).
so, some one related to this feild please let me know how to proceed

thanks for your suggesions in advance


#8959 does growing at 35c or 39c have an impact on ecoli growth ?

Posted by ravibiot on 28 November 2004 - 03:49 AM in Microbiology

yes, its true the variation in growth temparature has direct affect on the optimal growth,
what you can do is u do a series of growth experiments in different temparature inducbations with your strain of E.coli to know the optimal temparature.
best wishes

#8949 16S rDNA-RFLP

Posted by ravibiot on 27 November 2004 - 09:32 AM in Molecular Biology


i do investigation on microbial diversity, where, i used to amplify 16S rRNA gene which is of 1.5 kb using specific primers. To further proceed with DNA sequencing we have funding problem.
so, i have decided to see the diversity of microbes by restriction analysis of 16S rRNA gene.
but, i had a problem with the restriction analysis, where i get small 70 to 150 bp non specific bands (faint):::; along with the major banding pattern which corresponds (only major fragments corresponds for 1.5 kb) for 1.5 kb (that is my template 16S rDNA PCR product).
i had a doubt whether it's because of incomplete digestion and repeated the experiment with prolonged incubation. but, of no use.

so kindly suggest me how to proceed,

Thanks for your suggesions in advance


#8947 A Digestion problem

Posted by ravibiot on 27 November 2004 - 08:58 AM in Molecular Biology

"you have observed two bands of which one is of your interest
and the other of big size."

have u run your undigested template along with digestion product, to clarify whether any undigested template is left out in restriction digestion process. if so, please prolong the incubation time for complete digestion

good luck
ravi, INDIA

#8946 speckly DNA

Posted by ravibiot on 27 November 2004 - 08:49 AM in Molecular Biology

check the size of vector+target
and choose you need mini preps or maxi preps ext. kit
cood luck

#8944 Electroporator

Posted by ravibiot on 27 November 2004 - 08:16 AM in Molecular Biology

i recommend Biorads electroporator

#8943 Low copy number of plasmid

Posted by ravibiot on 27 November 2004 - 08:14 AM in Molecular Biology

1. u can try to grow cells in terrific broth
2. check the insert size limitations of the plamid
3. ensure your selction pressure conc. (antibiotic)


#8938 Need help with results of transformation...

Posted by ravibiot on 27 November 2004 - 05:34 AM in Microbiology

i suspect that, when the colonies arose from water trated samples, and you have obseved growth of mold its clear that the Amp plate you have used would be old and exposed much time to light which inactivated ampicillin.

#8937 Cellulase plate assay

Posted by ravibiot on 27 November 2004 - 05:12 AM in Microbiology


Does anyone know of another cellulase plate assay apart from using Congo Red? Thanks.

Cellulase utilization also can be checked on M9 mininal salts medium (Manitais et al. 1982. lab manual for gene cloning) amended with 0.9% of cellulose. incubate for 7 days and observe for halo around the bactirial colony indicated the production of cellulase.

#8694 Rhamnolipid production test

Posted by ravibiot on 17 November 2004 - 09:44 AM in Microbiology


i work on microbial degradation of hydrocarbons, its relevent to screen the degradative baceria for the production of rhamnolipids which reduces the surface tension in liquid culture medium.
i have a problem in the palte assay, wehre getting a blue colour zone formation around the colonies on SW blue agar (seigmund and wagner, 1991) which indicates positive reactrion. surprize is that, i did not get any blue zone for control strain also (Pseudomonas CHAO, CHAO660) which are reported for the same.

if some one can get me this article i am greatful for their help.
Siegmund, I., and Wagner, F. (1991) Biotechnology Techniques 5, 265-268

so, plese some one related to this field are highly appreciated for their help.

thank you in advance


#6551 PCR product purification

Posted by ravibiot on 12 August 2004 - 09:55 PM in PCR, RT-PCR and Real-Time PCR

hello gentle man,

i used to use promega whizard pDNA purification kit.

i hav used it to purify 256bp DNA product.. so no soubt to follow it.

u have to give more incubation time with the purification resin and hav to getly mix it for more than 5 min.

all the best

best regards


#6550 Minimal media

Posted by ravibiot on 12 August 2004 - 09:47 PM in Microbiology


welcome ur ideas and suggestions -

i work with isaolation of microbes potential for degrading various hydrocarbon pollutants. having a problem with salt precipitaion with minimal media (MM2, MSM).

request your suggetion.


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