PCR, Cloning, Restriction Digestion, Ligation, Transformation, Plasmid et al

• Papri Kundu

  I can't get induction of a previously induced protein using same induction procedure

  I have previously cloned, induced and standardized the method of purification of a membrane protein of 3kb but couldn't store it till now. I am now trying to induce the protein again using the sameI have previously cloned, induced and standardized the method of purification of a membrane protein of 3kb but couldn't store it till now. I am now trying to induce the protein again using the same plasmid (pET21b+gene of desire) transformed in E.coli BL21 but I can't get induction in the same conditions as previously. I have tried to double digest the plasmid containing the gene of desire with the restriction enzymes BamHI and XhoI by which the vector and the gene was double digested before ligation. The total plasmid (pET21b+gene of desire) was 8kb. The plasmid was not digested, therefore I tried to digest the plasmid from glycerol stock (pET21b+gene of desire in DH5a). The plasmid was also not digested. I tried with the glycerol stock of previously induced protein (pET21b+gene of desire in BL21). The digestion was successful and the bands of 3kb(gene)and 5.4kb(pET21b) was obtained but I couldn't get induction. I isolated the plasmid and transformed in fresh competent cell, but still couldn't get induction. I was inducing the full length protein of 99kDa at 298K for 8hrs at 2mM IPTG. Previously I have tried to induce it at 0.5-1mM IPTG but can't induce. In literature the truncated form of this protein was induced at 2mM IPTG 303K for 4hrs. In my case the induction was not much increased at 298K for 8hrs. The protein is a e.coli protein. Please help in troubleshooting.

  Recent replies ⋅ Show All (11)

  • 
    Ancy Thomas replied

    Dear We have some experiences with the pET series vectors and it sometimes show very in consistance in restriction digestion after storage in glycerol. You can check for the insert in your plasmid byDear We have some experiences with the pET series vectors and it sometimes show very in consistance in restriction digestion after storage in glycerol. You can check for the insert in your plasmid by PCR specific to your gene. It is observed that over a period of time, the T7 based E. coli vectors loss its transcriptional ability. This may be because of mutations in promoter region or transcription regulators. It is advisable to sub clone your gene into your vector of interest again which may solve all your problems related with protein expression. All the best.

    9 hours ago
• Cristine Menezes

  How can I study floral polymorphism into the same specie?

  I tought to study the genes expression, but there aren't nothing printed about which responsable genes or pathways metabolic for polymorphism of the colour and size flowers for this specie. SomeI tought to study the genes expression, but there aren't nothing printed about which responsable genes or pathways metabolic for polymorphism of the colour and size flowers for this specie. Some suggestion?

  Recent replies ⋅ Show All (4)

  • 
    Cristine Menezes replied

    Thanks!!! I'm reading now!

    18 hours ago
• Doctor Muhandas

  Labeling of southern probe by DIG PCR.

  I am trying to label DNA probe by DIG PCR. First I successfully optimized the amplification of gene by simple PCR reagents. Then I used DIG components for PCR reaction mix as recommended by Roche DIGI am trying to label DNA probe by DIG PCR. First I successfully optimized the amplification of gene by simple PCR reagents. Then I used DIG components for PCR reaction mix as recommended by Roche DIG manual including unlabelled DIG control and DIG label control provided with KIT. In addition to this I made separate reactions with normal amplicon master mix as the other control. I got bands of expected length with Unlabelled control (using kit material) and from normal amplicon master mix but there is no band from Labeled DIG reaction (gel pic is attached). What is the problem and how can I overcome this?

  

  Recent replies ⋅ Show All (7)

  • 
    Doctor Muhandas replied

    Thanks BH Kong for your suggestion.Its good to contact them for the problem. Thanks

    1 day ago
• Macarena Gomez-Lira

  Pseudogene EDNRA in gene expression anlysis

  Is the pseudogene for EDNRA, which does not contain a polyadenylation signal, a problem when analyzing EDNRA gene expression in melanoma cell lines? May I use random primers for cDNA synthesis?

  Recent replies ⋅ Show All (2)

  • 
    Macarena Gomez-Lira replied

    Thank you Ketty, I will contact him.

    2 days ago
• Kirsty Skinner

  Genomic DNA quantification

  I have a question regarding quantifying a genomic DNA sample. I extracted my genomic DNA using the Qiagen DNeasy kit, and a colleague suggested eluting the sample in water rather than the providedI have a question regarding quantifying a genomic DNA sample. I extracted my genomic DNA using the Qiagen DNeasy kit, and a colleague suggested eluting the sample in water rather than the provided buffer. Having done this I then attempted to quantify my DNA using a nanodrop. Readings appear normal, with the expected amount of DNA (ng/ul), however upon repeat readings of the same samples the variation is huge! The nanodrop appears to be in working order, after I checked using lambda DNA standards. Has anyone else experienced this or explain the variation that i'm seeing, as I cannot stanadardise my molecular work without knowning the original concentration of DNA. Thanks

  Recent replies ⋅ Show All (8)

  • 
    Zachary Bent replied

    I would suggest using a qubit (Invitrogen). It's cheaper than a nanodrop and gives much more consistent results.

    2 days ago
• Md. Nazmul Islam

  For animal study, lets say for seeing anticancer activity of a plant in vivo, how many minimum no of rats should be in a group?

  Will it be statistically explainable? For me my uni animal ethical committee approved only 5 for each group, will it be statistically explainable? I will have several groups with my therapeuticWill it be statistically explainable? For me my uni animal ethical committee approved only 5 for each group, will it be statistically explainable? I will have several groups with my therapeutic agent, placebo, carcinogenic agent etc. But if each of the group contains 5 subjects, is it okay?

  Recent replies ⋅ Show All (9)

  • 
    Edwin J Routledge replied

    As with any animal study it is vital to know the size of the effect you want to see, and take into account the variability you are likely to encounter between animals independent of treatment. PowerAs with any animal study it is vital to know the size of the effect you want to see, and take into account the variability you are likely to encounter between animals independent of treatment. Power analysis can help you determine the sample size needed. The best ethical argument is that if your experiment has insufficient power, you will not be able to prove anything staististically, and therefore the animals you have used are wasted. If power analysis suggests you can have 5 animals, then great. If not, make a robust statistical case for more. Good luck! Ed

    5 days ago
• Dipon Das

  I have a novel bacterial protein, which while isolating, doesn't come out in the supernatent as it forms inclusion bodies, thereby making it impossible to crystallize. Can anyone suggest what II have a novel bacterial protein, which while isolating, doesn't come out in the supernatent as it forms inclusion bodies, thereby making it impossible to crystallize. Can anyone suggest what I should do in order to characterize it and see its localization in bacteria? In silico characterization has been done, but it lacks significant homology.

  Recent replies ⋅ Show All (5)

  • 
    Birendra Singh replied

    why dont you refold the protein by dissolving inclusion bodies into urea/ Gu-HCl and successive dialysis or dilution refolding? If your protein do not lose its activity, e.g. if protein can refoldwhy dont you refold the protein by dissolving inclusion bodies into urea/ Gu-HCl and successive dialysis or dilution refolding? If your protein do not lose its activity, e.g. if protein can refold back correctly, you may get lots of protein from inclusion. On the other hand if yr protein is a membrane/ or membrane anchoring then you can truncate it to get soluble domain.

    5 days ago

• Post protected

  Join ResearchGate now to read this post.

  Recent replies ⋅ Show All (3)

  • 
    Rowena F Stern replied

    Research and Testing offer 454 sequencing for $200 per sample per 10000 reads or $150 for 3000

    6 days ago
• Md. Nazmul Islam

  I got band smearing in my agarose gel electrophoresis. The smear was quite strange where it stopped exactly at a specific bp.

  My PCR template was a PCR amplicon amplified from human DNA (nested PCR) which has been diluted 200 times. Taq buffer conc. = 1X, MgCl2 conc. = 2.5 mM, dNTP = 0.2 mM, Taq polymerase = 0.5 U. Four setMy PCR template was a PCR amplicon amplified from human DNA (nested PCR) which has been diluted 200 times. Taq buffer conc. = 1X, MgCl2 conc. = 2.5 mM, dNTP = 0.2 mM, Taq polymerase = 0.5 U. Four set of primers were used for a single reaction (multiplex PCR). As shown in the picture, the band with a green arrow was my intended band (960 bp). The smear was stopped exactly at 700 bp and the smear appeared as a 'ladder shape' with discrete bands. Adjusting the annealing temperature did not solve the smearing problem where every time I managed to make the smear gone, the intended band was also disappeared. What other parameter that I still can attempt to optimised? My DNA template is a GC-rich template. Do i have to increase the melting temperature? Thank you.

  

  Recent replies ⋅ Show All (1)

  • 
    Agnieszka Paulina Kijewska replied

    It looks like overload sample. The longest band is usually the most concentrated and smears during electrophoresis. Did U try to solve it for egzample 5 times and run again? BTW, look on the longestIt looks like overload sample. The longest band is usually the most concentrated and smears during electrophoresis. Did U try to solve it for egzample 5 times and run again? BTW, look on the longest band from marker: it's also smearing.

    6 days ago
• Elizabeth Hernandez

  Multiplexing Assays?

  Hello so i will be doing a multiplex assay real soon, but i am needing to order my primer/probes my question is how to determine what gene is most abundant in trying to multiplex? I have heard of theHello so i will be doing a multiplex assay real soon, but i am needing to order my primer/probes my question is how to determine what gene is most abundant in trying to multiplex? I have heard of the database NCBI blast but i am not aware of how to use it. if anyone can help it would be greatly appreciate it.

  Recent replies ⋅ Show All (1)

  • 
    James Borrone replied

    Your question is a bit nebulous - could you specifiy exactly what your concern is? The primary concern when multiplexing is whether the primers/probes you are using will either form primer dimersYour question is a bit nebulous - could you specifiy exactly what your concern is? The primary concern when multiplexing is whether the primers/probes you are using will either form primer dimers with one another, or amplify some non-target region(s).

    6 days ago
• Manik Vohra

  Molecular pathology (gene to disease and vice versa) - present day technologies and strategies?

  I have gone through some methods but I want to gather as much as I can so if someone can help me out with this I'd be grateful to them.

  Recent replies ⋅ Show All (1)

  • 
    Seema Trivedi replied

    Have you tried MIM morbid (OMIM morbid) database of genes and disease associations? Information on gene variations also give information about disease implications or predispositions. In differentHave you tried MIM morbid (OMIM morbid) database of genes and disease associations? Information on gene variations also give information about disease implications or predispositions. In different databases one can get information about gene variations.

    9 days ago
• Fatemeh Babaei

  Heart Tissue Lysis

  I want to lyse heart tissue, could anybody supply any useful protocols that work on my case?

  Recent replies ⋅ Show All (10)

  • 
    Vivekananda Gupta Sunkari replied

    Add some protease & phosphatase inhibitors......................PMSF, DTT, Sodium arthovenadate to ripe buffer and lyses the tissue.............good luck

    12 days ago
• Heverton Dutra

  Dosing DNA

  Can anyone please help me figuring this out. I was talking with some friends, analysing the DNA purity when dosing with nanodrop and they said that a good/pure DNA is present when we have a relationCan anyone please help me figuring this out. I was talking with some friends, analysing the DNA purity when dosing with nanodrop and they said that a good/pure DNA is present when we have a relation 260/280 around 1,8 and nothing too far from this number. Well, my point is: isn't 2+ a good relationship since 260nm stands for the DNA and 280nm stands for the aromatic ring present in tryptophan and tyrosine. so, for example if we have a score around 3, it means we have in general terms even more DNA than protein when compared to a 1,8 score. Thanks!

  Recent replies ⋅ Show All (7)

  • 
    Bindhya Sadhanandhan replied

    Values between 1.8-2.0 determines pure good quality DNA!!

    13 days ago
• Majid Tafrihi

  Does DMSO, when used as a solvent, effect gene expression (at protein level) in cell culture?

  I have treated my cells with two different drugs. One of these was disssolved in MeOH and the other in DMSO. In the control samples, we expected to see the same pattern (in western blotting), butI have treated my cells with two different drugs. One of these was disssolved in MeOH and the other in DMSO. In the control samples, we expected to see the same pattern (in western blotting), but they differ. Could anybody suggest a reason for this?

  Recent replies ⋅ Show All (12)

  • 
    Majid Tafrihi replied

    Dear Xiaojin Liu thanks for your comment. I agree with you and I have to test your idea and then change my solvent.

    15 days ago
• Lena Ilan

  CSF-1-dependent promoter

  For the positive control in my project I need a promoter that initiated by SCF-1/CSF-1R activation. Does anyone have in mind such an optimal promoter? Thank you.