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

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ascacioc on Tue Aug 28 20:20:32 2012 said:


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

Yes I do follow you but i must clear one confusion here ,From the colony that i plated after transformation into competent e.coli TOP10 i am doing my midid prep , right ..The vector keratinase has Hind111( 1383)restriction site and Sfil(361) that I could see from the information provided by the company IN THE PLASMID MAP

-siddharthsameer-

siddharthsameer on Wed Aug 29 07:41:04 2012 said:


ascacioc on Tue Aug 28 20:20:32 2012 said:


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

Yes I do follow you but i must clear one confusion here ,From the colony that i plated after transformation into competent e.coli TOP10 i am doing my midid prep , right ..The vector keratinase has Hind111( 1383)restriction site and Sfil(361) that I could see from the information provided by the company IN THE PLASMID MAP


Yes I do follow you but i must clear one confusion here ,From the colony that i plated after transformation into competent e.coli TOP10 i am doing my midid prep , right ..The vector keratinase has Hind111( 1383)restriction site and Sfil(361) that I could see from the information provided by the company IN THE PLASMID MAP: sO I THOUGHT IN ORDER TO CHECK AFTER THE MIDI PREP WHETHER I HAVE MY KERTINASE IN IT OR NOT I DIGESTED WITH HINDI 111...
abd as far as the vector is concerned my vector is pPICZalpha A which is 3,6kb and in the same way it was transformed in the competent cell and then it was plated on the Zeocin resistant marker....And from the colony that i got i am trying to do my Maxi prep.. and for the vector i tried with Ecor1 AND KPN1::
MYVECTOR IS NOT IN MY SEQUENCE(KERATINBASE) both are differently performed by me..
my main aim is to get the keratinase sequence in more concentration from midi prep and similarly vector in more concentration from maxi prep.. afetr i get these two i will start the pcr and everythng from scratch,. this is the main idea behind this...This was the idea my supervisor told me and m trying to iumplement it but unsuccesful... I hope everything is clear for this time.. i think you confused with the cloning, and offcourse its my fault of not making you clear....if u have more thngs to be asked kindly let me know i would be obliged and thankful to you..Now can u tell me why my midi and maxi prep is giving me the right result..
In between i would like to know one more thing is it possible to keep the preculture at 4c after attaining the od of 1 overnight and then use ir tommorow for making my main culture...my preculture has antibiotic..kindly let me knowww
regards

-siddharthsameer-

Yes it can be kept preculture at 4oC; I believe in everything fresh but I have seen people doing it all the times and not suffering because of this; you only have to take care that this culture will have a longer lag phase since the bacteria will need to 'wake up' their metabolism from the 4oC stage.

The rest comes soon.

Andreea

-ascacioc-

I do not understand why you want to get better concentrations from midi/maxi prep: you told me that you got 800 ng/uL for both of them. Indeed, you could get more considering what you are midi/maxi prepping. But we are not here in a contest of perfect prepping; 800 ng/uL is more than enough for your purposes aka cloning.

Now, since we are sure that we have this part straighten out. I want to tell you smth else: when you get the gene from the synthetic company, you do not have to check it for the insert; trust me: they make it their business to double and triple check that the insert is there (especially GeneArt); I usually take it directly for the double digest with the REs for cloning (but I order the gene flanked by the REs sequences I need for cloning) and then gel extraction. If you do not have the gene flanked by the needed REs you need to add them by PCR, see above how I do it. But for the sake of the check: the plasmid from GeneArt is 2300 bp + 1400 bp of your insert, when single digested should lead to a band of 3700 bp (in between the thick 3000 bp marker band and the one above it, which is 4000 bp). This is what you see in lanes 3, 4 and 5. So, your insert is there. Moreover, in the lane of non-digested insert you have 2 bands which is specific to plasmids (actually depending on the loaded concentration, you should have 3 bands) It is ok to have more than one band for a circular plasmid. This is expected. So everything is good here.

BTW: we call HindIII with 3 capital I which stands for roman number 3 (I was confused there for a second what you are talking about); unfortunately you cannot use HindIII for cloning in your vector; but you can use SfiI (just take care that you have everything in frame with the epitope and the alpha secretion factor in the end of the cloning, double and triple check this; most mistakes of why is not my protein expressing? come from here)

With the vector: I usually do not check that either (usually I know what I have in my collection); I do not do a restriction check; I double digest (as above) with the needed REs; for this I expect only one band of approximately the total length of the vector. Even with one RE you should see a similar one band. In the case of the EcoRI you have as expected the 3.3 kb band. However, in the case of the KpnI it looks like the undigested vector to me. I believe that it is more a problem of the KpnI than the vector. Now, I remember that once I also had a problem with the KpnI and either it was too old or I did not like the provider. Anyhow, I ordered it from another company and then it worked. But since then I do not use KpnI (Old Romanian saying: once burned with hot soup, I blow even in cold yoghurt). So, unless you need this KpnI for cloning, do not use it because it looks like it doesn't work for you.

To sum it up: your gel looks perfect. Everything is ok except your KpnI :)

Andreea

-ascacioc-

ascacioc on Wed Aug 29 09:37:08 2012 said:


I do not understand why you want to get better concentrations from midi/maxi prep: you told me that you got 800 ng/uL for both of them. Indeed, you could get more considering what you are midi/maxi prepping. But we are not here in a contest of perfect prepping; 800 ng/uL is more than enough for your purposes aka cloning.

Now, since we are sure that we have this part straighten out. I want to tell you smth else: when you get the gene from the synthetic company, you do not have to check it for the insert; trust me: they make it their business to double and triple check that the insert is there (especially GeneArt); I usually take it directly for the double digest with the REs for cloning (but I order the gene flanked by the REs sequences I need for cloning) and then gel extraction. If you do not have the gene flanked by the needed REs you need to add them by PCR, see above how I do it. But for the sake of the check: the plasmid from GeneArt is 2300 bp + 1400 bp of your insert, when single digested should lead to a band of 3700 bp (in between the thick 3000 bp marker band and the one above it, which is 4000 bp). This is what you see in lanes 3, 4 and 5. So, your insert is there. Moreover, in the lane of non-digested insert you have 2 bands which is specific to plasmids (actually depending on the loaded concentration, you should have 3 bands) It is ok to have more than one band for a circular plasmid. This is expected. So everything is good here.

BTW: we call HindIII with 3 capital I which stands for roman number 3 (I was confused there for a second what you are talking about); unfortunately you cannot use HindIII for cloning in your vector; but you can use SfiI (just take care that you have everything in frame with the epitope and the alpha secretion factor in the end of the cloning, double and triple check this; most mistakes of why is not my protein expressing? come from here)

With the vector: I usually do not check that either (usually I know what I have in my collection); I do not do a restriction check; I double digest (as above) with the needed REs; for this I expect only one band of approximately the total length of the vector. Even with one RE you should see a similar one band. In the case of the EcoRI you have as expected the 3.3 kb band. However, in the case of the KpnI it looks like the undigested vector to me. I believe that it is more a problem of the KpnI than the vector. Now, I remember that once I also had a problem with the KpnI and either it was too old or I did not like the provider. Anyhow, I ordered it from another company and then it worked. But since then I do not use KpnI (Old Romanian saying: once burned with hot soup, I blow even in cold yoghurt). So, unless you need this KpnI for cloning, do not use it because it looks like it doesn't work for you.

To sum it up: your gel looks perfect. Everything is ok except your KpnI

Andreea

thank so much i am feeling little bit relieved but still I am attaching the keratinase map that was sentr by the company kindly see to it and my gel and then let me know whether everythng is fine for my gel and my midi prep or not...
but i have one stupid confusion , since the hind iii cuts at 1013 as my supervisor gave me a chart where it was written.. so when i digested it with hindiii the band should come at there and for the undigested sequence it should be 1,4 kb....and similarly for my vector for undigested it should be 3,6kb..why is that for keratinase sequence and for my vector sequence all is in the same order.. kindly clear my doubt.
you are really helpful...
Attached File

-siddharthsameer-

Even if you cut at 1013, it does not matter: it is a circle that you cut at one position; no matter where this position is, the result is the same : one band of the total length of the circles circumference.

Now that I have seen your map: do not use SfiI for cloning; if you cut with SfiI from the GeneArt vector, you will have your insert but you will not be able to control the direction of insertion. So, use this plasmid from GeneArt as a template for your PCR and insert EcoRI site on the 5' end and XhoI or NotI at the 3'. Again: take care that your gene will be in frame with the rest of the tags, VERY-VERY IMPORTANT!!

Andreea

-ascacioc-

ascacioc on Wed Aug 29 11:03:01 2012 said:


Even if you cut at 1013, it does not matter: it is a circle that you cut at one position; no matter where this position is, the result is the same : one band of the total length of the circles circumference.

Now that I have seen your map: do not use SfiI for cloning; if you cut with SfiI from the GeneArt vector, you will have your insert but you will not be able to control the direction of insertion. So, use this plasmid from GeneArt as a template for your PCR and insert EcoRI site on the 5' end and XhoI or NotI at the 3'. Again: take care that your gene will be in frame with the rest of the tags, VERY-VERY IMPORTANT!!

Andreea

THANK You so much, now i am releved and now i can perform my rest of the experiment.....You are a saviour.. vielen dank :), any how i did my pcr today and tomorrow i will check on agarose... hope everythng is fine...and will let you know :)

-siddharthsameer-

I will post here what Sameer has written to me on the personal conversation and also try to answer it:
yesterday I did the pcr using my sequence that i had it from midi prep but Today wzhen i saw it it had different band of 2 kb but where i expected it to be of 1,4 kb.., i mad pcr of 4 samples that all had the same.. can you tell me waht could be the reason , is it due to any contamination or something like that....what should i do now.. kindly help me out..waiting for ur reply

Question: what are your primers specific to? I am assuming the gene from the start until its end, nothing more... Are you adding through tags any other sequences?
If I were you I would ignore it (it is not a big huge difference, it might be an artifact of the agarose; even though 600 bp it is a bit of too much of an artifact) and clone it and send it for sequencing to check what it is there. Or just send the PCR product for sequencing with the primers used for the PCR. Without seeing the primers and the plasmid sequence used for template, I cannot tell you more.

Andreea

-ascacioc-

Speaking about agarose artifacts aka DNA not running at the expected size:

http://www.protocol-online.org/forums/topic/26601-my-pcr-product-is-larger-than-expected/
http://www.protocol-online.org/forums/topic/19923-digested-pcr-product-migrate-slower-than-uncut/

-ascacioc-
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