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plasmid extraction troubleshooting,please help !!

PET21a

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9 replies to this topic

#1 M_shabani_medbiotech

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Posted 02 June 2013 - 09:07 AM

hi every one
i need help with plasmid extraction . my plasmid is PET21a and as u know it's an expression vector so it gives low concentrations in extraction ( or maybe i am the problem !! :D ) . any body has any suggestions to get higher yields ??
i truly appreciate ur help

#2 phage434

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Posted 02 June 2013 - 09:13 AM

We have no clue as to how you are doing it now. How could we tell you how to improve?

#3 M_shabani_medbiotech

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Posted 02 June 2013 - 09:17 AM

oh right ! i'm sorry :)
i do alkaline miniprep , u know the protocol , right ?

#4 phage434

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Posted 02 June 2013 - 09:21 AM

I know about 20 protocols, all of which are roughly categorized as "alkaline miniprep." Please tell us in detail what you are doing. How do you grow the cells? Culture medium? Antibiotics? What OD do you harvest at? What buffers are you using? What kit, if any? Columns? Did you add ethanol to the wash buffer?

All of these things make a difference, and could be the source of a problem.

#5 M_shabani_medbiotech

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Posted 02 June 2013 - 09:35 AM

here is how i do it ...

1. Grow culture overnight in 5 mL of LB with antibiotic for 12-16 hours at 37C with 180 rpm shaking ( ampicillin 100 microgram/microlitr)
2. centrifuge 5 mL for 5 minutes and remove all media
3. Resuspend pellet in 200 uL GTE and let sit for 15 minutes
4. Add 400 uL if fresh 0.2N NaOH/1%SDS, mix by inverting 6 times, and let sit for 5 min on ice
5. Add 300 uL Pottasium acetate, and mix by inverting 6 times and let sit on ice for another 5 min
6. Centrifuge for 10 minutes
7. Transfer supernatant to new tube and extract with an equal volume of choroform, phenol, isoamyl alcohol
8. Vortex and centrifuge for 5 minutes
9. Transfer supernatant to a new tube
10. Add 2 volumes of cold 95% EtOH and leavein -20 for at least 1 hour
11.Centrifuge for 15 minutes at 4C
12. Remove supernatan and drain tubes on a paper towel by propping them up on a sharpie to allow the EtOH to drain off
13. add 1 mL cold 70% EtOH, and centrifuge for 5 minutes
14. Remove supernatant, drain well
15. Resuspend in 50 uL of TE..

Edited by M_shabani_medbiotech, 02 June 2013 - 09:37 AM.


#6 M_shabani_medbiotech

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Posted 02 June 2013 - 09:40 AM

u know beside the low yield of plasmid concentration , another annoying problem i face is the huge amount of RNA even after Rnase treatment ! i would appreciate any kind of help with this problem too Posted Image

BTW , it's transformed into DH5a !

Edited by M_shabani_medbiotech, 02 June 2013 - 10:14 AM.


#7 phage434

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Posted 02 June 2013 - 11:22 AM

I think you really mean 100 ug/milliliter ampicillin.

You don't specify the concentration of potassium acetate -- Adding it should produce a white precipitate. The supernatent should have a pH of 5-6 or so.

What is the pH of the phenol-chloroform. Commonly this is prepared at two different pH's: 7.5 for DNA extraction, and 4.5 for RNA extraction. Make sure you have the correct version. Read this page on phenol:
http://openwetware.org/wiki/Phenol

I would usually do a chloroform only extraction at this point to remove phenol, but the ethanol precipitation probably does this job.

I would add at least 2.5 volumes of ethanol, preferably a bit more. This is the most likely fault I see in your protocol. The spin should be at high speed, and you should note the expected location of the pellet. Be careful in removing the supernatent to avoid accidentally removing the pellet, both here and in the 70% ethanol wash. This is an easy place to lose your DNA. You could add pellet-paint NF (Novagen) to visualize the pellet the first few times you do this.

You should dry your sample after the 70% ethanol wash similarly to the previous way, and test by smell to see if the ethanol is gone. Five minutes of the tube open on the bench is usually sufficient. Do not over dry, which will make the DNA hard to dissolve.

You may want to dissolve in less than 50 ul, to increase concentration. Make sure to vortex the tube after adding the TE, so that the pellet dissolves.

You can add RNAse A to the initial resuspension buffer to help remove excess RNA. Also, note the pH of the phenol/chloroform is correct. Read this page on composition of the Qiagen buffers, especially P1, their version of GET: http://openwetware.o.../Qiagen_Buffers

#8 M_shabani_medbiotech

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Posted 03 June 2013 - 06:18 AM

WOW ! u have A LOT of information !! :)
Thank u :)

potassim acetate is 3M . i don't know the PH of phenol chlorophorm . i should probably try 2.5 volumes of ethanol this time .

hey would u answer some other questions please ?
1. what exactly does potassium acetate do ?
2. what about i use isopropanol instead of ethanol ?

#9 phage434

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Posted 03 June 2013 - 06:42 AM

1. The potassium acetate does two things. First, it neutralizes the NaOH in the lysis buffer, bringing the pH back toward neutral or acidic. Second, the potassium ions form potassium dodecylsulfate, which is more insoluble than sodium dodecylsulfate. This precipitates a white material, which helps to trap the genomic DNA during the high speed spin which follows.

2. Isopropanol works fine for the DNA precipitation, but is much less volatile than ethanol. You can use smaller amounts of isopropanol -- 0.6 to 0.8 volumes instead of 2.5 volumes. This may result in larger amounts of salt precipitating. I would continue to use a 70% ethanol wash, which will evaporate more quickly, and will be better at removing the salt.

#10 mdfenko

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Posted 03 June 2013 - 10:14 AM

there's another thing that the potassium acetate does.

the potassium ion, in the presence of ethanol (or isopropanol) will ionically bond to the phosphate in the dna backbone and precipitate the dna.

sodium, ammonium or lithium acetate can also be used to precipitate dna but in your case, as phage434 says, the potassium will also precipitate the sds.

Edited by mdfenko, 03 June 2013 - 10:15 AM.

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