Hi everyone. Thankyou in advance. Actually I need large amount of plasmid for transfecting suspension cells using XLG protocol for Protein expression (structural biology experiments). The commercial Maxi-prep kits are quite expensive for such extensive plasmid use. So if there is any optimized protocol which you are already practicing in your lab for large scale plasmid purification will be of much help.
Hi there,
There are Alkaline lysis/Phenol extraction/Isopropanol precipitation protocols in the Maniatis book... You may find a recipe in the article below:
https://hal.archives-ouvertes.fr/hal-00575229/document
Dear Syed
a possibility is the tradizionale cscl ulttacentrifugation approach but i think that before move to in which is long and complex you can try to check for kit with better price or test if the coloumns of the kit are reusable to re-produce the same dna on the same coloumn and saving money just acquiring extra buffers.
However, What do you mean for high amount?
i'm working with expi293 cells (which are expensive but guaranteed amazing yields) to produce mgs of proteins which require 1ug dna/ml and in some (rare) cases to acheive good yields i growth Also up to 300-500ml of cells and generally i'm using omega Biotek midi kit which are not so expensive.
you can find some details about yields and costs on my blog
:https://proteocool.blogspot.com/
ProteoCools n12 at page 6
good luck
Manuele
Hi there,
There are Alkaline lysis/Phenol extraction/Isopropanol precipitation protocols in the Maniatis book... You may find a recipe in the article below:
https://hal.archives-ouvertes.fr/hal-00575229/document
I attached my protocol for the CsCl plasmid prep.
This works very well, and is "old school"
I have never had more plasmid DNA with such purity
with any kit.
Best Wishes!
https://mcardle.wisc.edu/sugden/protocols/Alkaline%20Lysis%20Plasmid%20Prep%20ver1.pdf
https://tools.thermofisher.com/content/sfs/brochures/D17309~.pdf
https://www.ars.usda.gov/northeast-area/docs/she/dna-extraction-by-cscl-ethidium-bromide-sops/
I haven't done a Cs Cl prep in many years. You can get beautiful DNA that way but the one thing that causes the most problems is dealing with the Et Bromide. You get a lot of waste and also contaminate a lot of things with it. You should also know that Chlorine bleach is not appropriate treatment for contaminated equipment. It causes damage the things you wash with it and it doesn't actually decontaminate. it increases the mutagenicity of EtBr. You need to have an appropriate activated carbon kit to remove EtBr from aqueous solutionns. See the link below, to Columbia University's EtBr cleanup protocol :
https://research.columbia.edu/sites/default/files/content/EHS/Policies/EthidiumBromidePolicy.pdf
Thankyou all. All these comments were of much help.
Manuele Martinelli I am using 293F cells but not able to get enough protein so am trying different methods. What method you are using to get considerable protein amount?
For me, am testing two methods:
1st Method:
> I seed around 0.7M cells/ml 24 hours prior to tansfection.
> Next day I transfer the transfection mix to the flask with continous shaking. DNA to PEI ratio (1:3)
> harvest cells after 48hours.
2nd Method.
> pellet down the cells i.e 20M/ml and add 50ug DNA/ml to it. Add 100ug PEI. Shake for 3 hours after transfection, then dilute the cells to 4 M cells/ml. Harvest after 48 hours.
What possible recommendations you want to suggest?
Thanks in advance.
Dear Syed
when you wrote: not able to hey enough, what do you mean? whixh are your yields?
i'm using expi293 which are an improved version of hek293f and i perform transfection with expifectamine that is more similar to lipofectamine that pei therefore is nor simple compare our protocool.
in our case i pellet down the cells, i resuspend it at 3M/mi and i perform transfection with 1ugdna/ml cells. i grow the cells at 37C - 120rpm and 8% of CO2, i add the enhancers after 24h and i collect the surnatant with the protein after 72h. i. this way using a pcdna 3.4 vector with kozac sequence and a signal peptide (generally l' testine 2-3 different signal peptide in parallel) i obtain very nice yields for the most of proteins. however nor all the protein express, my success rate actually is around 70% and domain desing in some cases is very critical.
probably before test expi293 you can try:
- try to use lipofectamine and Free style medium with your cells:
- try to review the clone sequence.
good luck
Manuele
Manuele Martinelli Thankyou for your kind suggestions. For me, 50ml transfection yield is less than 0.5 mg.
What additives do you use to enhance protein expression. I read about VPA and Sodium Butyrate that they enhance the yields upto six folds, but didn't try them yet.
Dear Syed
sincerelly i do not know which are the additives that are present in the expi293 medium to enanche protein expression but for some proteins we obtain amazing yields ( up to 10mg from 30ml of transfection) but there is variability.
Manuele
Dear Manuele Martinelli ,
I mean to say that as you said you add enhancers 24 hours post transfection. what are those enhancers? are they meant to enhance protein expression?
Regards
Dear Syed
the Thermo do not report the name of the 2 enhancers
in a their patent https://patentimages.storage.googleapis.com/39/56/04/8a266c4efa31a5/WO2013166339A1.pdf
at page 29 they report
"In some specific though non-limiting embodiments described below, "Enhancer 1" generally refers to 0.25 mM - 1 mM valproic acid, and "Enhancer 2" generally refers to 5 mM - 7 mM sodium propionate"
but sincerelly i'm not sure that are the components really used in the expi293 kit
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0007814_Expi293_ExpressionSystem_UG.pdf
Manuele
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