A platform to address questions regarding methods and development of new methods.

• Cunhua Shao

  How to make up the TAA solution?

  I want to develop a rat cirrhosis model using 0.03%TAA(thioacetamide) as the drinking water, but I am afraid of that the TAA will resolve, so what should i use to make up the TAA solution, tap waterI want to develop a rat cirrhosis model using 0.03%TAA(thioacetamide) as the drinking water, but I am afraid of that the TAA will resolve, so what should i use to make up the TAA solution, tap water (in China ), double-distilled water or saline solution ? Thank you !

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  Recent replies ⋅ Show All (23)

  • 
    Pascal Lopez replied

    Hi Joanna Sometime Qiagen preps give strange results concerning ratio of optic density. However, yours OD are quite good. But depending of what you want to do next, you may want cleaner plasmid. SoHi Joanna Sometime Qiagen preps give strange results concerning ratio of optic density. However, yours OD are quite good. But depending of what you want to do next, you may want cleaner plasmid. So you can reprecipitate your DNA in 2.5 vol of 100% ETOH and 0.1 vol of 5M NaCl. But this time, do it in a SMALL volume (I supose you are in about 200 µl of water or T10E1. So add 20 µl of 5M Nacl and 500 µl of 100% ETOH). Mix by hand until you see a nice DNA "pelote" pellet in the tube. No need for centrifugation. Pick the DNA pellet with the end of a yellow tip (just the pellet, do not aspirate the pellet, it should stick on your tips), put it carrefully in a new tube. Remove what is left of 100%ETOH (there is some in the pellet), rince the pellet in large volume of 70% ETOH (500µl to 1ml). Centrifuge and resuspend in water of another buffer. You plasmid shoud be cleaner. Good luck

    2 days ago
• Yang Pengbo

  Our early study validates that a protein is modified by SUMO-1 (human). How can I get the sumoylation site of the protein of interest using MS?

  The method is used to transplant the sumoylation pathway into Escherichia coli to purify sumoylated proteins directly from E. coli. The question is whether this method (co-transfection the MutualThe method is used to transplant the sumoylation pathway into Escherichia coli to purify sumoylated proteins directly from E. coli. The question is whether this method (co-transfection the Mutual SUMO-1[R95GG] and Substrate protein only) is suitable for eukaryotic cells? Thanks

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    Vladimir Barkhatov replied

    I think protection of Lys needs to receive same triptic fragments from native protein and its SUMOylated form, besause (as I know) SUMO must be binded to one of the Lys residues and most probablyI think protection of Lys needs to receive same triptic fragments from native protein and its SUMOylated form, besause (as I know) SUMO must be binded to one of the Lys residues and most probably protects this Lys from trypsinolysis. But it's a theory, practically it may occurs that not all potential trypsinolysis sites will be cleaved, and different in native and SYMOylated form, also it's some probability that trypsine can cleave SUMO-protein bind. Also if your protein has also glycosylation, it becomes harder to analyze spectra.

    4 days ago
• S. B.

  Experience with mRNA isolation kits?

  Hello, Does anyone have suggestions for mRNA isolation kits? What kits have you used and how was the quality? Any advice is appreciated.

  Recent replies ⋅ Show All (69)

  • 
    Sundararajan Jayaraman replied

    We routinely use TRIZol to extract total RNA from a variety of tissues including human peripheral blood mononuclear cells and mouse tissues. For microarray analysis Qiagen's RNeasy kit is needed forWe routinely use TRIZol to extract total RNA from a variety of tissues including human peripheral blood mononuclear cells and mouse tissues. For microarray analysis Qiagen's RNeasy kit is needed for purification. We have used kits from SA Biosciences, now Qiagen, for the purification of microRNAs from mouse tissues with success.

    5 days ago
• Itzel E Calleja-Macias

  Does anyone have a good protocol for migration assay using a transwell system?

  I'm working with Hela cells and we want to see if these cell migrate after the we treatment them.

  Recent replies ⋅ Show All (7)

  • 
    Georgina Coló replied

    Tray with 8 micron transwells costar, you could put the drug under the transwell, and then check the cells that crossed by flowcitometry. Or by chemotaxis chambers.

    9 days ago
• Dr. Bharat Bhusan Patnaik

  Double digestion of TOPO pCR 2.1 TA Cloning vector with the gene of interest not giving insert band of expected size

  My gene of interest (Partial N-terminal fragment) have been cloned in TOPO TA Cloning vector-Invitrogen. I want to express the gene in the expression pET vector and produce the recombinant protein.My gene of interest (Partial N-terminal fragment) have been cloned in TOPO TA Cloning vector-Invitrogen. I want to express the gene in the expression pET vector and produce the recombinant protein. After TOPO cloning, i have run PCR with expression primers having BamHI and HindIII sites and i got expected size that i have sequenced. The sequence is also matching to the original sequence. But when iam digesting the TOPO cloning vector with the insert with BamHI and HindIII, iam getting insert not of the expected size but 100bp more than the expected size. The unexpected size insert have been gel-purified and run on PCR with expression primers, in this case i get expected size results. Why this anomaly. Please suggest.

  Recent replies ⋅ Show All (12)

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    Dr. Bharat Bhusan Patnaik replied

    Thanks once again. Dear Dr. Halgand, PCR with the ligation product (pET vector with insert) is to check the sucess of the ligation product. For this iam using pET vector forward and reverse primerThanks once again. Dear Dr. Halgand, PCR with the ligation product (pET vector with insert) is to check the sucess of the ligation product. For this iam using pET vector forward and reverse primer (T7). Alternatively we can use vector forward primer(T7) and gene specific reverse primer or gene specific forward primer and vector reverse primer (T7). This i hope can validate our construct towards transformation. I need to ask though, that as i will be verifying my reading frame by sequencing, is it better to transform into non-expression host as DH5-alpha and select for the white colonies as pET lacks lacZ gene for blue-white colonies. After confirming the sequence, transformation can be conducted in a expression host as BL21-DE3 cell lines and the target protein can be optimized by IPTG induction. Please comment. Dear Dr. George, i have tried sequential double digestion as well with NEB enzymes (BamHI and HindIII) apart from fermentas enzymes, but i could not get my insert at the expected size, though PCR of the insert with expression primers gave the expected size. Do you suggest for overnight digestion.

    9 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.

  

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• Yoshus Hoosier

  XbaI/SpeI Digestion

  Has anyone ever done this digestion. The digestion efficiency seems very low. The plasmid is very bright and the insert looks very faint on the gel? There is no dam methylation problem btw. AnyoneHas anyone ever done this digestion. The digestion efficiency seems very low. The plasmid is very bright and the insert looks very faint on the gel? There is no dam methylation problem btw. Anyone know what the issue is?

  Recent replies ⋅ Show All (8)

  • 
    Rolf-Peter Ryseck replied

    I wouldn't expect any problem (and had none) using buffer 2 or buffer 4 and Biolab enzymes. However, careful with XbaI as overlapping dam methylation TCTAGAtc methylation prevents digestion. If youI wouldn't expect any problem (and had none) using buffer 2 or buffer 4 and Biolab enzymes. However, careful with XbaI as overlapping dam methylation TCTAGAtc methylation prevents digestion. If you know the sequence, check it and regrow plasmid in dam-negative E. coli strain, if needed. Otherwise, one of your enzymes might simply be dead.

    10 days ago
• Andrea Leishman

  RNA extraction - Sarkosyl

  I wish to use phenol:chloroform extraction for the isolation of total RNA from murine spleen cells. The protocols I have read all include Sarkosyl in the denaturing buffer. Can this detergent beI wish to use phenol:chloroform extraction for the isolation of total RNA from murine spleen cells. The protocols I have read all include Sarkosyl in the denaturing buffer. Can this detergent be replaced with any other more common detergents? The extract will be used for downstream PCR reactions. Thanks.

  Recent replies ⋅ Show All (2)

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    Andrea Leishman replied

    I have just realised that the protocol(s) I have been looking at are an updated version this protocol. I am still not clear on the exact function of the Sarkosyl in the lysis buffer, but it seemsI have just realised that the protocol(s) I have been looking at are an updated version this protocol. I am still not clear on the exact function of the Sarkosyl in the lysis buffer, but it seems that it may be unnecessary.

    12 days ago
• Madhusmita Borah

  Specific media for isolation of acidophilic bacteria

  Hi Can anyone please help me and let me know about media commonly used for isolating acidophilic bacteria (pH <=2)?
• Sandip De

  Sonication standardization problem for HCHO-crosslinked CNS and discs collected from third instar larvae using Bioruptor.

  I have been trying to standardize sonication protocol for HCHO-crosslinked CNS and discs collected from third instar larvae of Drosophila using Bioruptor. I have used discs and CNS from 10 larvae, II have been trying to standardize sonication protocol for HCHO-crosslinked CNS and discs collected from third instar larvae of Drosophila using Bioruptor. I have used discs and CNS from 10 larvae, I do find variations in fragments sizes (either 200-600 bp or 500-1500 bp) using same setting and same reagents. I tried changing several factors e.g. sonication tube, buffer, number of cells, even used different bioruptor. Surprisingly all the samples of each sonication batch produces similar sizes of DNA fragments which cancels the possibility of improper lysis of discs and CNS (I do crush the crosslinked discs and CNS using plastic pestle for eppendorf tubes). Please tell me if you have any idea of whats going on, I have been trying to standardize this for last two months. Thanks...
• Madeleine White

  Does anyone have a good protocol for the isolation and culture of mouse liver leukocytes?

  I would like to be able to get a reasonably pure culture of immune cells isolated from the liver so I can look at cytokine response and flow cytometry. Cheers!

  Recent replies ⋅ Show All (1)

  • 
    Vincent Vercruysse replied

    There are 2 attachments First step , for non parenchymal cells obtention second step: for cells of interest ( choose a markers for your leukocytes) Best regard

    gentleMACS-Liver-04.pdf

    14 days ago
• Jordi M. Argilaguet

  Spleenocytes proliferation and cell death.

  When doinf CFSE-proliferation assay with spleen cells from mice I get more then 60% of cell death after 3 days, specially in non-stimulated wells. Same protocol works perfect with PBMCs from humansWhen doinf CFSE-proliferation assay with spleen cells from mice I get more then 60% of cell death after 3 days, specially in non-stimulated wells. Same protocol works perfect with PBMCs from humans (20% of cell death). Can anyone help me? Thanks in advance

  Recent replies ⋅ Show All (10)

  • 
    Juan M Zapata replied

    Koshika is absolutely right. Adding 50 microM beta-ME to the cultures of mouse lymphocytes is a must if you want to keep cells from dying.

    14 days ago
• Paulina Troncoso Escudero

  Has someone a good and easy protocol to purify vesicles from astrocytes?

  I need to purify intracellular vesicles and analyze their content.
• Louise Chopinet

  Nucleus purification/isolation protocols?

  Does anyone have something to share? I have already made some research on the internet, but I found only very old publications. Thank you!

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