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SDS can precipitate out of solutions at RT, so if buffers that do contain SDS have a whitish precipitate in them, some people can mistake if for contamination.
#148235 what is the contamination in buffers
Posted
on 17 January 2013 - 03:52 AM
#148066 No activity of expressed protein obtained from pET32a cloned gene
Posted
on 14 January 2013 - 10:59 PM
The protein I am working with goes into insoluble fraction on expression. I need protein in soluble form for its activity assay. The Trx-tag is generally use to get the protein into soluble fraction. As Thioredoxin (Trx) itself is highly soluble protein, it brings any protein in-frame to it in soluble fraction. Later, that tag can be cut from the target protein using enterokinase. thus Trx is not a tag for purification of protein but in enhances the solubility of it. For purification I use His-tag.
#148055 pEGFP C-1/N-1 Cloning
Posted
on 14 January 2013 - 06:58 PM
I would never directly clone into target vector. I always clone to pJET first, and then subclone. usually my PCR product ends don't digest well, so we follow this strategy.
#148024 Wet transfer of proteins between 100-200kDa
Posted
on 14 January 2013 - 12:05 PM
Low and slow is always best for a wet transfer. I use 15-30 V overnight. Higher voltages cause problems with heating. Minimize the amount of methanol you use and you can add some SDS to increase mobility.
#147984 pET 32 (+)
Posted
on 13 January 2013 - 01:50 PM
It refers to the orientation of the multiple cloning site of the vector.
#147824 Delivery to cytosol of cultured cells
Posted
on 10 January 2013 - 10:39 AM
A comparison of the delivery of Atto488-bovine serum albumin into cultured human sarcoma cells by nine different commercial delivery products, with an exploration of a photochemical technique enhancing endosomal escape:
Mellert K, Lamla M, Scheffzek K, Wittig R, Kaufmann D. Enhancing endosomal escape of transduced proteins by photochemical internalisation. PLoS One. 2012;7(12):e52473. doi: 10.1371/journal.pone.0052473. Epub 2012 Dec 21.
http://www.plosone.o...al.pone.0052473
Mellert K, Lamla M, Scheffzek K, Wittig R, Kaufmann D. Enhancing endosomal escape of transduced proteins by photochemical internalisation. PLoS One. 2012;7(12):e52473. doi: 10.1371/journal.pone.0052473. Epub 2012 Dec 21.
http://www.plosone.o...al.pone.0052473
#147891 pEGFP C-1/N-1 Cloning
Posted
on 12 January 2013 - 05:05 AM
can you do a double digest to re-confirm your insert is there? I have worked with N2 and N3, and your protocol looks fine to me. One thing that I suspect is that your insert itself could be toxic? or your competent cells are not viable enough due to a possible break down of your freezer. Trust me this has happened to us like more than 5-6 times that during weekend we had no electricity and nobody knew about it. These cells might grow in broth again, but are not suitable for transformation anymore. Kan plates also last for long time, unlike amp plates that can't be used after a month.
#147707 Dam Dcm Negative Strain
Posted
on 08 January 2013 - 06:27 PM
You can use strains of bugs without the methyl transferases. These strains will be Dam-/Dcm-. I don't know of any way to do this post extraction if that is what you are asking.
#147923 TAP Water
Posted
on 12 January 2013 - 02:04 PM
Depends very much on which depth you want to analyse it in and what equipment and possibilities you have. Is it for something like a microbiology class assignment or do you want to perform a serious check ?
http://en.wikipedia...._water_analysis
http://www.epa.state...rt/microman.pdf
http://en.wikipedia...._water_analysis
http://www.epa.state...rt/microman.pdf
#147798 Large-Scale Purification of Non-Secreted Protein in E.Coli or CHO Cells
Posted
on 09 January 2013 - 09:36 PM
Alert: Thread hijack ahead 
Actually, both are acceptable. Usually one would use lower case, but litre is the exception, where upper case can be used when concerned about mixing up a 1 and an l. And also, most countries spell it "litre". I'm pretty sure it's is just the Americans who use "liter".
(source: Internationional bureau for weights and measurements http://www.bipm.org/...apter5/5-1.html)
Curtis, on 09 January 2013 - 08:24 PM, said:
We write liter with capital L. Like mL, uL.
Actually, both are acceptable. Usually one would use lower case, but litre is the exception, where upper case can be used when concerned about mixing up a 1 and an l. And also, most countries spell it "litre". I'm pretty sure it's is just the Americans who use "liter".
(source: Internationional bureau for weights and measurements http://www.bipm.org/...apter5/5-1.html)
#147795 Large-Scale Purification of Non-Secreted Protein in E.Coli or CHO Cells
Posted
on 09 January 2013 - 08:24 PM
We write liter with capital L. Like mL, uL.
Of course there are other methods, some people prefer to not use detergent, and instead break cells physically. there are machines for that. But in the lab most people use lysis buffers, because they are cheap. RIPA is really strong, and honestly it is not my favorite either. I used CHAPS buffer for some time, and then for my fractionation studies I used digitonin buffer. You can study different types of detergents at Wikipedia. I'd recommend you to study them carefully, it's important to know their differences.
1000 L is very large volume though, it's like 1 ton. it's a pilot scale. Even if you use lysis buffer you need to centrifuge that 1000 L to get a clear supernatant. that would need a huge centrifuge. I would also think for an alternative if I were you. I will check this and let you know because I have never lysed this many cells.
Of course there are other methods, some people prefer to not use detergent, and instead break cells physically. there are machines for that. But in the lab most people use lysis buffers, because they are cheap. RIPA is really strong, and honestly it is not my favorite either. I used CHAPS buffer for some time, and then for my fractionation studies I used digitonin buffer. You can study different types of detergents at Wikipedia. I'd recommend you to study them carefully, it's important to know their differences.
1000 L is very large volume though, it's like 1 ton. it's a pilot scale. Even if you use lysis buffer you need to centrifuge that 1000 L to get a clear supernatant. that would need a huge centrifuge. I would also think for an alternative if I were you. I will check this and let you know because I have never lysed this many cells.
#147719 Insulin production
Posted
on 08 January 2013 - 09:12 PM
I don't know how much info you already have, they do this by cloning the two isoforms and transforming into e.coli. Perhaps you can check this animation:
http://www.abpischoo...tion_allTopic=1
If you need more in-depth material you need to check the Diabetes Journal under American Diabetes Association.
http://www.abpischoo...tion_allTopic=1
If you need more in-depth material you need to check the Diabetes Journal under American Diabetes Association.
#147644 Replication of the Plasmid DNA
Posted
on 07 January 2013 - 02:37 PM
I don't know what you mean by a big deal, but if you were referring to my reluctance to give a full answer - I do that so as to make you think, it helps you learn and to become a better scientist...
#147661 Restriction Enzymes
Posted
on 08 January 2013 - 02:00 AM
Pangea, on 08 January 2013 - 01:56 AM, said:
Ok , lets say i want gene sequence of an RE from IMAGE consortium. And produce my own RE in E.coli. I am no considering the cost and sso on.. . Question is if its practcally possible in the LAB.
sure.
Its possible but not practical
You simply need the DNA sequence of the restriction enzym with the promoter etc... there is no reason that it would not work, but if you have to collect the enzyme/purify it etc.. it will take a lot of time, money ...
BTW: check this topic http://www.protocol-...ing-polymerase/
its not about a RE , but the same goes for you. Altough, your would be harder to collect/purify
#147559 How to transform adherent cells to suspension (CHO cells)
Posted
on 06 January 2013 - 10:07 AM
There is a wealth of good information on the web about adapting CHO adherent cells to suspension culture and serum free medium, search for "adapting CHO adherent cells to suspension". You can find information from Invitrogen/Gibco,Thermo Scientific Hyclone, and other suppliers of
SFM... about adaption methods as well.
SFM... about adaption methods as well.
