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plasmid extraction


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#1 hidayahwannur

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Posted 08 February 2012 - 07:58 AM

i used traditional method alkaline lysis to extract plasmid, but i still cant get the band when i run agarose gel electrophoresis. these what i have done:

10 ml culture cell + kanamycin overnight at 37C
observe grow or not
transfer into falcon tube, centrifuge 13000 rpm, at 4C, 10 min
remove supernatant
add 100ul alkaline lysis solution 1, mix the pellet
transfer into apendorf tube
put on ice
add 200 ul solution 2,invert 6 times, put on ice 3-5 min
add 200 ul solution 3, invert
centrifuge 5 min, 4C, 13ooo rpm
transfer supernatant into new tube
add 5 ul RNAse, put in water bath , 10 min at 37C
add phenol ,same volume with supernatant
centrifuge 5 min
transfer the upper layer into new tube
add chloroform , centrifuge, transfer the clear solution into fresh tube (wash 3 times with chloroform)
add 2 volume of alcohol 95%, centrifuge, remove supernatant
add 70%alcohol , centrifuge
remove supernatant, dry overnight
add eluent buffer, store at -21C

#2 HOYAJM

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Posted 08 February 2012 - 10:48 AM

What does the growth look like after the overnight incubation? If it is not as dense as it should be, you could try using a different media like S.O.C Broth. It appears that you are following a well established protocol, so I dont think it is that. My last suggestion would be to try to see if you have a Qiagen Miniprep Kit around and use it.

#3 bob1

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Posted 08 February 2012 - 03:48 PM

Make sure your solution 2 (NaOH/SDS) is fresh - it absorbs CO2 from the atmosphere and stops working.

#4 Sandy143

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Posted 08 February 2012 - 07:59 PM

Hi,

I use promega kit for plasmid extraction but the sequence results are very dissapointing ~ 40 clones were sent for sequencing and only 2 clones hit the target organism (is this normal?). Sometimes I got low yield of plasmid (~ 60 ng/ul). I'm doing immunoscreening of cDNA library looking for potential clones against parasitic disease. Once the potential clones (phage) were obtained, I do in vivo excision to convert the phage into plasmid followed by plasmid extraction prior to sequencing. These clones are quite specific since I'm testing them with panel of serum samples, so I expect to get high chances of good clones sequenced and hit the target organism.

Does anyone has idea where should I start trobleshooting?
Thanks

#5 hidayahwannur

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Posted 08 February 2012 - 11:02 PM

today i did the plasmid extraction again. i have 4 samples, and below was the result:

sample plasmid DNA concentration(ul/ml) purity A 260/280

1 98.5 1.90
2 10877.5 1.96
3 376.9 1.92
4 62.5 1.86

is that ok?

#6 scolix

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Posted 09 February 2012 - 10:25 AM

it seems, ok. But do you see it on the gel?
I am assuming, its ug/ml not ul/ml.
The second sample has a lot of DNA. A bit too high for miniprep.

#7 hidayahwannur

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Posted 09 February 2012 - 05:49 PM




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625]
Posted Today, 02:25 AM


it seems, ok. But do you see it on the gel?
I am assuming, its ug/ml not ul/ml.

[/center]
The second sample has a lot of DNA. A bit too high for miniprep.

i m sorry..yes, ug/ml..not ul/ml. but i cant see it on the gel. but i will repeat it again. maybe due to lack of skill. can u give some tips for me on agarose gel electrophoresis? what precaution should be consider? i use EtBr.

#8 HOYAJM

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Posted 09 February 2012 - 07:57 PM

today i did the plasmid extraction again. i have 4 samples, and below was the result:

sample plasmid DNA concentration(ul/ml) purity A 260/280

1 98.5 1.90
2 10877.5 1.96
3 376.9 1.92
4 62.5 1.86

is that ok?



I agree that the 2nd concentration is way too high. Even the third is higher than normal. Generally, around 50-200 ng/ul for a miniprep would be normal. Regardless, you should see SOMETHING on the gel when you run it.

So, from your miniprep, mix 2-10 ul with loading dye and run it on a 1.0% agarose gel with EtBr. Use TAE buffer and run for an hour or so. If what you have from that miniprep is DNA, you will see something, maybe a smear, hopefully a band. It only takes about 20 ng of DNA to be visible on a gel.

#9 hidayahwannur

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Posted 10 February 2012 - 10:05 AM

usually, what is the best ratio of the sample: loading dye? is that ok if i mix 5ul sample + 2ul loading dye?

#10 scolix

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Posted 10 February 2012 - 10:29 AM

It depends on the concentration of loading dye. Usually we have a 10x stock, so for a 20ul, add 18ul sample + 2 ul loading dye

#11 Ikar

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Posted 10 February 2012 - 10:51 AM

I have another question I am curious about: You let the pellet dry over night. Then it might be very very dry for sure. I usually use a speed vac for a couple of minutes (if I do not have much time) or let it dry for 1-2 h.

What are the consequences if there is a tiny leftover of ethanol? Why is it considered as so bad?

#12 HOYAJM

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Posted 10 February 2012 - 12:13 PM

According to Qiagen (from the miniprep handbook), the main consequence of leftover ethanol is that it can interfere with future enzymatic reactions that the DNA will be used in.

#13 Ikar

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Posted 10 February 2012 - 12:29 PM

But it is not mentioned which ones in particular, like restrictions and PCR reactions, or both?

#14 scolix

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Posted 10 February 2012 - 10:15 PM

Any reaction where enzymes will be involved, can be affected with ethanol. So both restriction digests and pcr.




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