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isolation of plasmid (midi and maxi prep) - (Aug/24/2012 )

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hello all
I did my Midi and maxi prep and this time the concentration and mass were also good, but when I did the restriction digestion with the restriction enzyme to check my band I could not see the band at the desired position.. it is showing something else...M really tensed and I dont know wat to do...
I did the single restriction digestion with ECOR1 and KPN1 for the volume og 50Microlitre containg 1 microlitre of my dna. and incubated for 2 hrs at 37C and then deactivated at 65C for 20min...
I am really clueless wat is going for...and for the control I added 1microlitre of my DNA with 19 micro litre of water..but on the agarose for my midi prep(Keratinase sequence)and Maxi Prep(Vector) is showing the same band...
I run the agarose at 70v 85mamp for 2hrs...
I really feel like crying,,, Kindly tell me what to do, should I make the restriction again on monday, and also check with the PCR about my sequence...
please give me some suggestion, m stuck here for 3 weeks..
regards

-siddharthsameer-

can you attach a picture of the gel? What concentrations of DNA did you get for the vector and the insert?

Andreea

-ascacioc-

Did you run intact plasmid on gel? check your natural plasmid with same size other natural plasmid by running on agariose. they should give band at almost same position. Have you tried O/N digestion?

-Inbox-

Hi i can attach the picture once i go to lab, but the concentration of my insert (1,4kb) is 872,3µg/ml (from midi prep)and for the vector (3,6kb) is 860µg/ml (from maxiprep)..
Now what should i do. i am doing the pcr for my sequence to check it and again the restriction digestion to check once again.. if the result is the same what shoul I do? kindly guide me, i am cluless...
regards


ascacioc on Sat Aug 25 06:45:52 2012 said:


can you attach a picture of the gel? What concentrations of DNA did you get for the vector and the insert?

Andreea

-siddharthsameer-

Concentration of the insert looks a bit too much for my taste (is it from a PCR? I remember you were cleaning up a PCR last week) Somehow, in your protocol described here I do not see you getting rid of the template for your PCR at any step.

Putting just 1 uL of your DNA in 50 uL of water for the digestion is too diluted. I usually have inserts 30-80 ng/uL and vectors 100-300 ng/uL (=ug/mL) and make restriction reactions of 20-30 uL 2h at 37oC (if I am in a hurry) but most times I do it overnight. The golden rule is not to have more than 20% of the volume restriction enzyme (due to the glycerol they are kept in, it is not nice to have too viscous samples, enzymes do not work when they are slowed down by slime:) )

So, to round it in a nice protocol for you, I will tell you how I am doing stuff:
-PCR the template with overhangs for restriction enzymes +1-2 more base pairs outside the restriction sites (you need some space for the enzyme to have where to sit) (you can find my protocol here: http://www.protocol-online.org/forums/topic/26610-primer-annealing-temperatures/ (it is one of the posts on the first page of the discussion)
-since you PCR the insert most probably using a plasmid as a template, you want to get rid of the template afterwards; there are two ways to do so, either run an agarose gel and do gel extraction (most people do so) but I am more of a conservative when it comes to losing parts of the insert (by gel extraction you always lose some of your DNA). This is why, after the PCR, I pool down the entire PCR corresponding to my insert (~70 uL) and add 1 ul DpnI (from NEB, I only use NEB enzymes; Fermentas are a bit crapy; they do not invest so much in their R&D; not as much as NEB does at least). DpnI digests only methylated DNA, which means that would digest the template plasmid (since this was amplified in bacteria, it is methylated while your DNA amplified through PCR is not). I incubate ON at 37oC, in the morning I inactivate the enzyme
- next I do a DNA clean-up (the entire 70 uL on a silica column) and elute in 15 uL MilliQ water (I usually use MilliQ water which was autoclaved as PCR grade water; it is important that you also rinse very well the bottle you are gonna use; you do not know who made Mg/Mn salts before you in that bottle and the dish washer did not wash it properly; I pre-incubate the bottle 3-5 times with MilliQ water and then discard before I use this bottle the first time and then for the rest of my project in that lab use the same bottle over and over again). I always elute from the mini prep (silica) columns in MilliQ water because I prefer not to have too many ions extra for the next reactions
Note: I always wash 3 times with NT3 when I do the PCR clean-up or gel extraction because you get contaminants from NT buffer that will screw you following reactions; check the 230/260 ratio at nanodrop when you measure concentration; high 230 absorbance is due to these contaminants
-restrict digest this insert (50-80 ng/uL) in a total volume of 20 uL (15 uL PCRed insert, 1 uL restriction enzyme 1, 1 uL restriction enzyme 2, 2 uL buffer appropriate for both enzymes; check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp#.UDtZpaMvmSo and 1 uL water or BSA (in case is required) --> 2h or overnight at 37 oC
-inactivate the restriction enzymes for 20 min and do a PCR clean up, elute in 15 uL PCR grade water (as above) and measure the concentration by nanodrop; I usually get 30-80 ng/uL; check the 230/260 ratio as well
This was for the insert. With the vector there are fewer steps. I usually miniprep my vectors from 6 mL of E coli grown 18-20 h in ZYM505 (special media for mini preps from Studier's autoinduction media article) (lyse bacteria from 3 mL ON culture in one eppi; per culture I use 2 lysis reactions and I pool them on one column silica column after clearing the lysate) and after the entire procedure I elute in 50 uL PCR grade water. I usually get 100-300 ng/uL (even though I got also 50 or 1000 ng/uL but these are extremes) but this might not work for everybody because some expression vectors lead to low concentrations especially if you use LB instead of ZYM505 (ZYM505 is quite difficult to prepare anyhow and if you do not have the needed components in your lab; forget about it; LB works just fine). To make long story short: I take 24 uL of 100-300 ng/uL DNA (take care that having too much DNA might inhibit your reaction; so dilute your DNA down to this concentration) + 1.5 ul of restriction enzyme 1 + 1.5 uL of restriction enzyme 2 + 3 uL buffer (as above) + the rest up to 30 uL total reaction, either BSA or water according to what is needed for the respective enzymes. Incubate 2h-ON at 37oC.
Note: I always dilute the 100xBSA down to 10-30x BSA in PCR grade water before I add it because I do not like pippeting less than 1 uL quantities unless I really need to and it is not crucial to have exact quantaties.
Note 2: I always add the enzymes the last thing in the reaction after everything else, including buffer is already there.
-inactivate the enzymes 20 min and run an agarose gel with your restriction digest of the vector (I usually put scotch tape on the comb to unite more wells; 2 are enough for 30 uL+loading dye). The trick to get good yields from gel extraction is to pour a very thin agarose; the thinnest you can get, 0.5 cm if you are good enough; the more agarose you have, the more DNA you lose); do the gel extraction with the extra NT3 buffer washes (I use the Macharey & Nagel gel extraction kit, former Nucleospin, Extract II); elute in 25 uL PCR grade water
-measure the nanodrop concentration of the vector

This is enough for your problem now, the rest will follow shortly :) It is like a TV series with episodes... stay tuned. :)

Andreea

-ascacioc-

BTW: if you have questions, do not be ashamed to ask; nobody was born knowing all these stuff; I also have learned them from my master thesis supervisor; but I had a very good supervisor, not like you :)

-ascacioc-

helloo here is the agarose gel of my midi and maxi prep.. the marker is of neb 1kb ladder...
the molecular weight of the marker is from 0,5 to 10 kb.. and just after the marker i have keratinase sequence, which is the keratinase that had the vector backbone of pMK-RQ(kanR) which was ordered from the company and then at the time of ligation experiment this sequence was transformed using e.coli competent cell, and the colony that i got from the agar plate (which was kenamycin resistant) i am trying to do the midi prep from that colony, but i am unsuccesful in getting my sequence.. today i performed again I got the same result ....
similarly for the vector..
i feel like crying, what should i do now, i cant proceed my experiment withou this....and the vector was also prepared on the zeocin plate and i am trying to do the maxi prep fromn that again
My keratinase sequence is 1,4kb and the vector is 3,6kb (pPICZalpha A) but on the band everythng is of same size..
Attached Image

-siddharthsameer-

on the undigested insert I see 2 bands; the upper band looks to me like the plasmid template band that you did not purify away. I gave you above two possible ways to get rid of the template: either DpnI digest (my favourite one) or a gel extraction before the restriction digest.

-ascacioc-

I really do not know what other things you do wrong. Read my protocol above + ligation protocol below and see what I do different and you have also some explanations why some steps must be included.

More, are you sure your PCR is successful? I mean, where is your 1.4 kb band? I see only bigger bands. Or are you doing RE from the plasmid from the company?

There is another possibility for you having the same bands for both vector and insert: contamination in one of the solutions with DNA from somewhere else; it happened once to me with my PCR water (embarrassing, I know ). I had a bit of plasmid from one previous cloning in my water and somehow I transformed it with my next cloning and I amplified it up to the point of seeing it only in the sequencing (coincidentally both genes were cloned in the same vector, same size, only different sequence).

Ligation: in a total reaction volume of 10 uL, take 80 ng insert (total amount DNA not 80 ng/uL) and 20 ng vector (both insert and vector from above); 1 uL 10X T4 DNA ligase buffer (NEB) (take care: this contains ATP that is destroyed in repeated freeze-thaw cycles; do not use very old buffer); 0.5 uL T4 DNA ligase (NEB) and top it with water up to 10 uL; 1 h at room temperature and transform 4 uL (chemical competent cells or 2 uL for electrocompetent cells) of this into DH5alpha, XL1Blue or any other cloning strain. Plate 100 and 900 uL of this transformant on appropriate antibiotic agar plates and wait a min of 16 h. Keep rest of ligation in the fridge for another trial if you have too many colonies after this.

Next day I do a colony PCR of 8-10 colonies from this transformation and then miniprep and send for sequencing. Tell me if you have also problems from then onwards. You told me that you do not have too much guidance in your lab, so I am willing to share detailed protocols.

Really, besides what I said above, I cannot see another mistake. Try to follow my way of doing and, worst case scenario repeat it from the beginning with new ingredients.

Good luck,

Andreea

-ascacioc-

In the light of new info I received from you through private messages (I am summarizing here the info needed for everyone to follow our conversation):
Lane2- marker
lane 3- keratinase sequence digested by hindi 3
lane 4 and 5- keratinase digested by hpa1
lane 6 and 7- Vector digested by ecor1
lane 8 and lane 9- vecor digested by kpn1
lane 11 undigested keratinase
lane 12- undigested vector
then the marker and again the undigested keratinase and undigested vector
My keratinase has to be 1,4kb and the vector to be 3,6kb
is not my pcr product.. its my sequence that was ordered form the comopany and this sequence was transformed into the competent e.coli cell and then plated on kenamycin plate and the colonies that we got i am trying to do the midi prep from these<...>
anyhow i tried myself doing the colony pcr and then i made the preculture and then I will try to do it again the midi prep....

Ok. So you did not PCR your insert as I assumed from your previous questions last week.

I have more questions:
why are you digesting the plasmid with the insert from the company with one RE at a time?
why are you digesting your vector with different REs than your insert?

When I order a gene from synthetic company I order it flanked by the REs I need for cloning. Otherwise I PCR the insert with primers that would add the needed REs. Then, I restrict digest both the plasmid from the company and the vector I am cloning it into with both REs (same REs) in the same time (2h or ON). I would expect for this restriction digest 2 bands (for both the insert containing plasmid and the vector).

By cutting your plasmid with one RE at a time you expect only one band, which obviously will be different than the size of your gene. Imagine a circle you cut only once: how many fragments do you get? Your plasmid from the company contains the plasmid backbone and your insert , all in a circle. You have to cut it twice to remove the insert. Do you follow me?

Andreea

-ascacioc-
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