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Sequential restriction digestion of plasmid


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#46 shimshady

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Posted 05 March 2009 - 11:20 AM

Back to my orignal question can you see plasmid DNA seperation at 1% agarose? I am trying to run just the plasmid itself and only see one band. Shouldnt i see 3? I see virtually the same band for digested and undigested plasmid. Any thoughts/?

#47 friendly

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Posted 05 March 2009 - 10:37 PM

The inserts look perfectly prepared. Cloning them into a standard high copy number vector, such as pUC19 should be straightforward from here. You will get many clones, each containing thousands of bases of DNA sequence from the banana genome. You can sequence some of these. I still have not heard what the goal of doing so is. This is surely not the way, for example, to completely sequence the banana genome, but that might not be what you want to do.


Thanks Phage for the reply.
The photo I show u in lane 3 and 4 is DNA from the Dragon fruit. Apparently I didn't know what is the genome size for Dragon fruits so will u suggest me to continue? Will it be a full sequence. ? I doubt that because nobody have discover the total genomic size of it.
My goal is to construct a genomic library first before doing further action on it. Might need to look for functional gene from the library.

please reply and thanks again.

#48 friendly

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Posted 10 March 2009 - 09:03 AM

any idea phage?
I have been wondering should I continue as the Kit for Library is costly ..

#49 phage434

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Posted 10 March 2009 - 12:28 PM

Why do you need a kit? The cost of a kit will be very low compared to the cost of sequencing any substantial number of clones. Where are you going with this? What is the end goal?

#50 perneseblue

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Posted 10 March 2009 - 01:10 PM

This is surely not the way, for example, to completely sequence the banana genome, but that might not be what you want to do.


I have to agree with phage434. This is most certainly not the way to construct a library.With fragment sizes that small (several kb), your library will required a hundreds of thousands of colonies to have a minimum of 3x coverage. If you are gene hunting, eukaryotes have introns, lots of them which can be really big. Fragments sizes of kb may not be able to able to capture your gene.

A cDNA library would probably be of more us. What is the project's objective?

If you are building a genomic library, it would be better if you used BAC to carry inserts 50kb to 150kb size range. Then your library need only contain several tens of thousands of colonies to have minimum coverage. You will need to do a partial digest on your genomic DNA. Then run out the partially digested DNA using pulse field gel electroporation.

(Do note that Ecoli doesn't like certain sequences. And if you are using restriction digest to fragment your DNA, DNA that doesn't have the restriction site will be under represented as ligated molecules and thus absent from the library.)

As for the Dragon Fruit... well as Phage434 has been saying, what is your purpose? If you are gene hunting, you could continue. Just screen a large number of colonies. With sufficient brute force and ignorance, you should be able to find your gene.

As phage434 has mentioned, you don't need a kit. Just the right vector. And screening this big library and maintaining it (if required) would be a lot more costly than the kit.
May your PCR products be long, your protocols short and your boss on holiday

#51 OA17

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Posted 01 May 2009 - 04:56 AM

To prevent religated vector you should Cip or Sap treat the vector. After your digestions are done, just add one ul of Cip/Sap enzyme to the buffer already present. Incubate at 37 degree for one hour and continue with purification and ligation. This removes the 5' phosphate which is critical for ligation. Now, the vector can only ligate with the insert since the insert has a 5' phosphate after digestion.


I thought of using Cip or Sap, but my supervisor does not provide me with these two. What is the difference between Cip or Sap? :huh:


Hi!

If your supervisor does not provide you with CIP or SAP, there is something else you can do to avoid the religation of the vector: just cut with a third enzyme in the middle of the fragment that you want to remove from the vector (an enzyme that should have a unique restriction site just between the other two enzymes that you are using for cloning). This might help you.

Good luck!

#52 liweixie

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Posted 21 June 2009 - 09:37 AM

To prevent religated vector you should Cip or Sap treat the vector. After your digestions are done, just add one ul of Cip/Sap enzyme to the buffer already present. Incubate at 37 degree for one hour and continue with purification and ligation. This removes the 5' phosphate which is critical for ligation. Now, the vector can only ligate with the insert since the insert has a 5' phosphate after digestion.


I thought of using Cip or Sap, but my supervisor does not provide me with these two. What is the difference between Cip or Sap? :)


Hi!

If your supervisor does not provide you with CIP or SAP, there is something else you can do to avoid the religation of the vector: just cut with a third enzyme in the middle of the fragment that you want to remove from the vector (an enzyme that should have a unique restriction site just between the other two enzymes that you are using for cloning). This might help you.

Good luck!

Hi QA17, you mean cut the vector with one enzyme where it is between the two cutting site. and then cut with the other two enzyme where you want to insert the PCR product into.
I had similar problem that I am working on construct plasmid. Using promega or fermentas company's enzyme, to cut the vector, most of time I got a lot single cut. i guess due to only 27 nt between two cutting site, the digestion efficiency is not too high.
One more question, I also use the same enzymes to digest the PCR product, 870bp. after ligation and transformation, I found almost all the PCR product was cut once.
Thank you!

#53 ampelo

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Posted 01 June 2011 - 12:23 PM

no, you do not need to purify the linear plasmid after the first digest. NdeI like NEB buffer4. And BamHI can work in buffer 4. Thus the sequential digest begins with NdeI. Once the DNA is linearised, add BamHI and continue the digestion.

An uncut preparation of plasmid DNA contains plasmids in several topologies. cut linear DNA, supercoiled circular DNA and nicked open circular DNA. Supercoiled DNA moves faster than linear DNA as it is more compact. Then comes cut linear DNA (due to shearing). And the slowest of all is the nicked open circular DNA, which has lost its twist and is now a floppy loop. A big circle has greater difficulties migrating through the agarose mesh.

Thus circular DNA doesn't run like linear DNA. And as the DNA ladders are linear DNA, you don't get the expected size.

If you are unfamiliar of how circular DNA looks, I suggest a sample of uncut DNA be run as control.

http://humgen.wustl....age/plsmid3.gif

Very good explanation. I have seen many people instead of giving clear explanation confuses even more! thanks.

#54 vinee2

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Posted 02 March 2012 - 08:02 AM

I do EcoR1 and BamH1 double digestion in buffer 3 for pET28a and also having the problem of self ligation...can anybody suggest if sequential digestion will be of any help...

#55 phage434

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Posted 03 March 2012 - 08:09 AM

This is unlikely to help. You can solve excess background by using PCR instead of cutting the plasmid with REs. Design primers with your desired cut sites and about 6 bp of additional junk 5' of the cut site, binding to your plasmid surrounding the MCS. Use minimal amounts of plasmid template. PCR amplify, purify (important!) then digest with your REs (in your case BamHI and EcoRI) plus DpnI. The DpnI cuts the circular plasmid,the BamHI and EcoRI cut the linear PCR product, leaving your linear, non-transformable DNA fragment.

#56 VJbiofication

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Posted 22 June 2012 - 02:13 AM

Hello people,

I just started to learn cloning. As I know, we incorporate same restriction sites at both ends of cDNA and digest with the one enzyme.
Can you tell me why do we need to do double digestion of cDNA? How to add 2 restriction sites in the same cDNA?
What is the use/application?

and the next question is: If we use double digestion in cDNA, how the plasmid will be manipulated? I mean, do we have to do the same double digestion with plasmids? Because we need to add both plasmids and cDNA to have the same cohesive ends. How/where a double digested cDNA will be in the final plasmid vector?

My questions may seem amateurish, but please help me with my questions. Or at least give me some links where I can read about this.

Thanks.
VJ.

#57 phage434

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Posted 22 June 2012 - 03:51 AM

You can clone with the same RE site on both ends of your construct, but it has several disadvantages. First, you have no control over the orientation of the insert -- it can go in forwards or backwards. Second, since you cut the vector only once, the vector can easily (and favorably!) religate rather than ligating your insert. This can sometimes be controlled by phosphate treatment of the vector after cutting, but this is (in my opinion) inferior in every way to using distinct enzymes at each end of your insert. The two enzymes must leave incompatible overhangs (that is, they cannot be isoschizimers). I'd recommend choosing enzymes that can be heat killed, avoiding a cleanup after RE digestion. The two RE digestions can be performed simultaneously in the same reaction in most cases (and you should choose enzymes that allow this). Most NEB enzymes now cut in buffer 4, for example.

#58 VJbiofication

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Posted 22 June 2012 - 07:12 AM

You can clone with the same RE site on both ends of your construct, but it has several disadvantages. First, you have no control over the orientation of the insert -- it can go in forwards or backwards. Second, since you cut the vector only once, the vector can easily (and favorably!) religate rather than ligating your insert. This can sometimes be controlled by phosphate treatment of the vector after cutting, but this is (in my opinion) inferior in every way to using distinct enzymes at each end of your insert. The two enzymes must leave incompatible overhangs (that is, they cannot be isoschizimers). I'd recommend choosing enzymes that can be heat killed, avoiding a cleanup after RE digestion. The two RE digestions can be performed simultaneously in the same reaction in most cases (and you should choose enzymes that allow this). Most NEB enzymes now cut in buffer 4, for example.


That is a very good explanation. I don't know why, but I didn't come across this kind of information in standard materials. Thank you.

I have another question: So as you said we have to have 2 different restriction sites on both flanks of the insert. So when I prepare/order the cDNA should I also include these sequences in the final insert: restriction site-start codon-insert-stop codon-restriction site?? Or how is it done? Please explain.

#59 mahrak

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Posted 23 June 2012 - 06:19 AM

I was just wondering, how about using more than two enzymes in a single reaction, say, ApaI, XhoI and EcoRI , would it be any different, or as long as the glycerol's kept in range it would work just as well as sweet, old double digestion? how about fermentas's Fast Digestion buffer and enzymes? has any one tried a triple digestion with them?




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