Molecular Cloning

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2 Ligation Procedure.
By Sahil Patel · Directorate of Groundnut Research

Daniel Cohen · University of Pennsylvania

Different temperatures are used for blunt end vs sticky end ligations. For blunt end ligations, there is no Watson-Crick base pairing to promote DNA f... [more]

Different temperatures are used for blunt end vs sticky end ligations. For blunt end ligations, there is no Watson-Crick base pairing to promote DNA fragment association. This renders the reaction less favorable in thermodynamic terms because there is no enthalpic (bonding energy gain) to offset the entropic cost of fixing the orientation and position of the two DNA fragment required to ligate two DNA fragments. By lowering the temperature (usually 14C, although some folks use 16C or even 4C), you can lower this entropic barrier by decreasing diffusional rates of the DNA. As Sezer points out, ligase activity is decreased at 4C, so there is a tradeoff between favorable energetics and decreased catalytic rate of the DNA ligase (thus necessitating longer incubations). In practice then, it is a matter of convenience whether one chooses 4C overnight, or a few hours at 14 or 16C.

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6 How can i transform a vector into Bacillus?
By Thanasan Nilsu · Chulabhorn Graduate Institute

Sabine Brantl · Friedrich-Schiller-Universität Jena

You should grow B. subtiils in Spizizen minimal medium (see other comments above) till onset of stationary phase (OD approx. 1.8 - 2.1), then wait 1.5... [more]

You should grow B. subtiils in Spizizen minimal medium (see other comments above) till onset of stationary phase (OD approx. 1.8 - 2.1), then wait 1.5. - 2 hr (competence is high for 2-3 hrs, then decreases again) and then use 0.8 -1 ml cells and add your DNA) For plasmid transformation, you need at least trimeric plasmids. It is not sufficient to linerarize plasmids, you should cleave your plasmid with an enzyme that has only a unique restriction site, and afterwards religate the mixture at high DNA concentration in a small volume to to obtain trimers or higher oligomers (check in 1 % agarose gels), otherwise the transformation efficiency is very low, as you usually have only a small percentage of trimers in your plasmid preparations.

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27 E coli colonies not growing in culture (again)
By Alice Dawson · University of Dundee

Pham Tuan · Nara Institute of Science and Technology

I agreed that the problem in this case may be resulted from toxicity of the inserted genes or recombinant proteins if you could make sure that your tr... [more]

I agreed that the problem in this case may be resulted from toxicity of the inserted genes or recombinant proteins if you could make sure that your transformants are exactly carrying recombinant plasmids by colony PCR.

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3 Convenient one-step cloning kit for sequencing?
By Asaad Azarnezhad · Tehran University of Medical Sciences

Ponnusamy Sasikumar · Madurai Kamaraj University

if you synthesis your PCR product by Taq DNA polymerase, i suggest you to use InsTAcloning-PCR cloning kit from Fermentas and it also cost effective.

if you synthesis your PCR product by Taq DNA polymerase, i suggest you to use InsTAcloning-PCR cloning kit from Fermentas and it also cost effective.

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5 Free software or website similar to vector NTI?
By Imran Khan · University of Incheon

Tushar Dutta · Indian Agricultural Research Institute

serial cloner, snapgene viewer, pDRAW32, DNA strider, plasmapper etc. but the thing is most of them are available as trial version for 1 month.

serial cloner, snapgene viewer, pDRAW32, DNA strider, plasmapper etc. but the thing is most of them are available as trial version for 1 month.

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21 Cloning a C-terminal GFP fusion: is it okay that I still have the start codon for both my gene and the GFP?
By Valerie Osterberg · Oregon Health and Science University

Fathia Soudy · Huazhong Agricultural University

Hi Valerie About your question is very good to ask it Because I have met a similar situation before in my experiment . I agree with David Chiluiza... [more]

Hi Valerie About your question is very good to ask it Because I have met a similar situation before in my experiment . I agree with David Chiluiza`s answer that your gene and GFP, are in a frame. My experiment was rescue experiment and GFP expression is very important to me because I found my gene already has expressed in the model system but GFP not and I changed to RFP and BFP and the problem was still with me so I sent my plasmid construction to campany to give me sequencing and my seq results is right so I changed my Splicing Factors that I used it with GFP (at first time I used Sl-1d) to Sl-2d :: GFP and my GFP already has expressed and finished my experiment.

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15 What's the reason of antibiotic resistance? When the plasmid is not exists
By Mohammad Jafari · Ferdowsi University Of Mashhad

Balajee Somalinga · University of Texas Southwestern Medical Center

Mohammad, Here are some suggestions Try plating a control LBA4404 on Kan + plates with out any transformation. a) If you see any colonies, then your... [more]

Mohammad, Here are some suggestions Try plating a control LBA4404 on Kan + plates with out any transformation. a) If you see any colonies, then your stock of LBA4404 already could contain a Kan resistance gene b) If don't see any colonies, your plasmid could be low copy number, so your yield could be low. In such a case, follow instructions for low copy plasmid purification such as larger culture volume etc. Good Luck, Balajee

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4 Is there any probability to show a fragment size 2300 bp being increased to more than 3000 bp after ligation?
By Hesham Abdullah · University of Massachusetts Amherst

Daniel Cohen · University of Pennsylvania

As Phoebe points out, Apa I is sensitive to methylation (dcm). Check for CCAGG and CCTGG sequences flanking your ApaI site in the plant-based expressi... [more]

As Phoebe points out, Apa I is sensitive to methylation (dcm). Check for CCAGG and CCTGG sequences flanking your ApaI site in the plant-based expression vector.

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14 Cloning mouse TH promoter in a luciferase reporter vector
By Valerio Piscopo · IGB - Institute of Genetics and Biophysics - CNR

Valerio Piscopo · IGB - Institute of Genetics and Biophysics - CNR

Tank You

Tank You

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7 Are my ligation conditions ok?
By Azam Safary · Kashan University of Medical Sciences and Health Services

Daniel Cohen · University of Pennsylvania

I guess it boils down to a question of how many colonies one wants to screen. When cloning works well, checking 2-3 colonies is sufficient. Lucas has ... [more]

I guess it boils down to a question of how many colonies one wants to screen. When cloning works well, checking 2-3 colonies is sufficient. Lucas has a decent point that in some cases you can brute force screen dozens of colonies to get a single correct one. Whether this approach makes sense for you may depend on how many colonies you have already, or are willing to screen.

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2 Megaprimer Problem
By Mohd Khairul Hakimi · Universiti Teknologi Malaysia

Gary Laco · The Roskamp Institute

I use the megaprimer method a lot and it works great for me, you can get your mutant in <2 hr (but still have to digest and clone it). I would double ... [more]

I use the megaprimer method a lot and it works great for me, you can get your mutant in <2 hr (but still have to digest and clone it). I would double check your megaprimer to make sure it binds 5'-3' on the template DNA to pair with the outside primer, and that your outside primer also binds in the correct orientation on the template DNA to make the correct size DNA product. Also, I always try to make the 3' end of the primers (the end that gets extended) to be in a GC rich region, that will make sure it binds tightly and gets extended properly. It is important to also make sure the megaprimer and outside primer have a similar GC content (important for the annealing step), and that you have a denaturation step of ~15-30 sec. at 95oC to make sure the megaprimer melts (time depends on the megaprimer Tm). The other issue is your primers, you have tested one set to make the megaprimer (so they work), but I would also test the two outside primers to see if they can amplify the correct "final" size 606 bp DNA (of course there will be no mutation in that test). Let us know if you make any progress.

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11 How can I get a eukaryotic protein in soluble form which was expressed in E.coli?
By Manjushree Mk

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4 What is the best method to identify Kd (dissociation constant) and Ka (association or equilibrium constant)?
By Thang Pham · Ho Chi Minh City University of Science

Andreas Brachner · Medical University of Vienna

Hi Thang, the problem is, if you detect with an antibody it's not quantitative anymore. The big advantage of radioactive labelling is its sensitivity ... [more]

Hi Thang, the problem is, if you detect with an antibody it's not quantitative anymore. The big advantage of radioactive labelling is its sensitivity and it allows quantitation via a scintillation counter (-> for determining the Kd value). The amount of 35S-methionin that is used in such assays is very little, and 35-S is also not so critical as other isotopes because it is easy to shield (its mainly beta irradiation) - but of course you have to have a special training to handle radioactive material. You may google if there's an alternative labelling technique available (via luminescence probably?) which allows a quantitative readout. Best wishes, Andreas

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52 Can anyone suggest companies that sell TA cloning vectors?
By Kashif shamim · Goa University

Sudip Das · Universität Würzburg

Invitrogen TOPO TA cloning are the best!

Invitrogen TOPO TA cloning are the best!

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3 In a luciferase reporter/pre-miR co-transfection experiment,
By Enrica Fabbri · Universita degli studi di Ferrara

Daniel Cohen · University of Pennsylvania

Enrica- I would suggest a constant level of luciferase plasmid (usually 1.5-2 micrograms per 6 well) and titrating your miR mimetic to demonstrate a d... [more]

Enrica- I would suggest a constant level of luciferase plasmid (usually 1.5-2 micrograms per 6 well) and titrating your miR mimetic to demonstrate a dose response. The range of miR mimetic doses should be based on how much you are using in other experiments to generate a phenotypic response or a change in expression of an endogenous gene target. HTH.

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25 When is it necessary to purify your DNA during cloning?
By Sarah Fazal · Kent State University

Wu Yang · Chinese Academy of Sciences

you can make the last step of gel purify together in one Spin Column (the ratio of the vector-instert can be roughly judged from the gel brand ).

you can make the last step of gel purify together in one Spin Column (the ratio of the vector-instert can be roughly judged from the gel brand ).

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1 No digestion plasmid DNA with BclI restriction enzyme?
By Anton Cheremnykh · State Research Institute of Genetics and Selection of Industrial Microorganisms

Daniel Cohen · University of Pennsylvania

Maybe there is not a BclI site in your vector, either because the plasmid map is wrong, the site was destroyed during cloning, or you did not isolate ... [more]

Maybe there is not a BclI site in your vector, either because the plasmid map is wrong, the site was destroyed during cloning, or you did not isolate the correct plasmid. You should try to run undigested plasmid to get a sense of DNA quality (is there RNA contamination?, is there any evidence of plasmid degradation prior to digestion?). Also, run a control digestion with other robust enzymes. e.g. HindIII or EcoRI, if present in your vector, to make sure the restriction mapping is consistent with your plasmid map.

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5 how can I isolate DNA from agarose gel with centrifuge method without remaning agarose with extracted DNA?
By Afrooz Rashnonejad · Ege University

Koen Venken · Baylor College of Medicine

I you just try to spin fluid with DNA from the agarose, a short spin (30 seconds) should work fine. I even made my own columns by putting some glass w... [more]

I you just try to spin fluid with DNA from the agarose, a short spin (30 seconds) should work fine. I even made my own columns by putting some glass wool in an eppy with hole made by needle on top of another eppy.

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28 Problem with separating linearized/circular plasmids for cloning (or problem with CIP maybe)?
By Johannes D.

Robert Marano · University of Western Australia

Most people seem to be concentrating on the digestion, CiP process, ligation etc. However, I think the opinions by Pablo Becker and Matthew Moreno are... [more]

Most people seem to be concentrating on the digestion, CiP process, ligation etc. However, I think the opinions by Pablo Becker and Matthew Moreno are probably the best and easiest to discount. If there are the same number of colonies on each plate then it would seem that antibiotic selection is not functioning. Were the plates made recently, was the Amp relatively fresh or had it undergone a number of freeze thaw cycles etc. When adding the Amp do you add to the melted agar or spread on top. If added, and the agar is too hot it will deactivate the Amp. As suggested earlier, try plating just the DH5 alpha with no plasmid

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11 How to draw a circular genome graph from a draft bacterial genome?
By Diogo Proença · University of Coimbra

Roy Ronen · University of California, San Diego

Hello Diogo, You can also try Sibelia at http://etool.me/software/sibelia It will automatically (and quickly) align the genomes and output a circula... [more]

Hello Diogo, You can also try Sibelia at http://etool.me/software/sibelia It will automatically (and quickly) align the genomes and output a circular genome graph (in both the circos and in interactive-html format). Good luck!!

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16 Any suggestions for establishing a Tetracycline inducible mammalian cell line using the TRex system from Invitrogen?
By Virginia Aragon · Georgetown University

Mohsen Basiri · Tarbiat Modares University

Thank you David I think you are right, repeated transfections may not be helpful in long term, though it can increase TR copies.. But I thing the leve... [more]

Thank you David I think you are right, repeated transfections may not be helpful in long term, though it can increase TR copies.. But I thing the level of TR expression is still the most important factor. Invitrogen has improved TRex system by using Flp-In Cell lines to avoid positional inactivation of TR and GOI. If the TR+cells which can tolerate higher concentrations of blasticidin are selected, they may contain TR insertions in active chromatin positions. So possibly express more TR too! Any way... I'm not sure!

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2 Can we use pCDH (plasmid) for simple transfection without packaging it into a lentiviral system?
By Muhammad Imran · Ajou University

Muhammad Imran · Ajou University

Danial Cohen....OK got it..Thanx for information....

Danial Cohen....OK got it..Thanx for information....

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5 pGEX-2T and Rosetta 2.
By Jihui Zhang · St George's, University of London

Jihui Zhang · St George's, University of London

Thanks everyone again! I have tried the above suggestions, here are the results and new problem: 1) From SDS-PAGE, it sounds there is band which the... [more]

Thanks everyone again! I have tried the above suggestions, here are the results and new problem: 1) From SDS-PAGE, it sounds there is band which the size is equivalent to the target protein. However it can be seen in the negative un-induced samples (no IPTG added) but the density is slightly lower. I wonder if I need to optimize the medium, e.g adding glucose etc to repress the expression. Does anyone can suggest a good idea please? Many thanks for your help.

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19 Transformation efficacy
By Christopher Bachran · National Institutes of Health

Koen Venken · Baylor College of Medicine

You want to keep the volume of DNA less than 10% of the volume of the cells. Preferentially as little as possible.

You want to keep the volume of DNA less than 10% of the volume of the cells. Preferentially as little as possible.

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2 How to generate a gfp_shRNA/Hek293 cell line?
By Subhanita Ghosh · International Centre for Genetic Engineering and Biotechnology

Daniel Cohen · University of Pennsylvania

pGIPZ is another good option.

pGIPZ is another good option.

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13 Colony PCR
By Anwar Khan · University of the Punjab

Abdul Hameed · University of Karachi

Try colony PCR with lesser amount of colony/DNA, lesser amplification cycles and a final extension of about 3-5min

Try colony PCR with lesser amount of colony/DNA, lesser amplification cycles and a final extension of about 3-5min

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8 Why do my pictures of large sequencing polyacrylamide gels come out blurry?
By Max Bobrovskyy · University of Illinois, Urbana-Champaign

Erika Norabuena · Mount Holyoke College

Hi, what % gel are you using? maybe you can increase your percentage so your samples diffuse a bit slower and that will help you to get a better bands... [more]

Hi, what % gel are you using? maybe you can increase your percentage so your samples diffuse a bit slower and that will help you to get a better bands resolution. Also, for how long are you exposing your gels? I have been running 7% polyacrylamide gels for DNAse 1 footprinting. The top part of the gel looks really good and my bands are sharp but the bottom part looks blurry. I usually leave it at RT (which is 22C) for 3 days because my counts (I use 32P) are very low. A few days ago I run another gel this time I prepared an 8% instead and let it at 4C for 3 days. This time my bands at the bottom looked less blurry but afterall it improved. Also, maybe after imaging your gel you could try to play around with a photo software that can help diminish the blurriness.

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2 How can I find DNA sequence of flp gene (for site specific recombination) in a commercial vector to express in Cyanobacteria?
By Khan Rezaul · Shanghai Jiao Tong University

Estanis Navarro · IDIBELL Bellvitge Biomedical Research Institute

You can try to clone the flp cDNA (it is intronless).

You can try to clone the flp cDNA (it is intronless).

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13 Transfection
By Leo Pham · Ajou University

Leo Pham · Ajou University

Thank you all for suggestions. I used Renilla vector as dual luciferase assay and it worked well.

Thank you all for suggestions. I used Renilla vector as dual luciferase assay and it worked well.

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