blunt-end cloning

[lwong]

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Hey yall! It's my first summer doing undergrad research, so don't laugh at me if my question is actually a very easy ques...lol.

I am planning to do blunt-end cloning. My question is how do you identify those that have the foreign gene inserted into the direction you want (I have a specific end that I want near the promoter)? Furthermore, how do you even isolate those plasmids that have the gene inserted in the desired direction?

Any resources (sites, books, etc) that you would recommend?

Thanks!

 

[Doctor&Geek]

With blunt-end or otherwise nondirectional cloning, you transform and pick up colonies as usual, but when you do a check restriction enzyme digest, you make a strategy which utilizees some restriction enzyme site within your foreign gene and one outside of your foreign gene to determine directionality.

An example:

--|--|-----|-------------------|-----
EcoRI SmaI XhoI SmaI

VERSUS

--|--|--------------------|----|-----
EcoRI SmaI XhoI SmaI

Your gene is cloned into SmaI. Lets say that if the orientation of your clone is correct, XhoI is at the 5' end of your insert. Therefore if you cut with EcoRI outside of the foreign gene and XhoI inside your gene, inserts in the correct orientation will be shorter than the ones in the wrong orientation. Having no insert at all would be obvious, and having multiple inserts may indicate multiple blunt end foreign genes being ligated.

Blunt end ligations are less efficient in general than sticky end ligations, and I suggest that you have a 3-5 to 1 ratio of insert to plasmid when ligating.

Final note: make sure you ligate at 4 degrees, rather than 14 degrees overnight.

I recommend either blue book (Molecular Cloning) or red book (Current Protocols).

 

[PostalWookie]

Make sure you use CIP or SAP to dephosphorylate the vector 5' ends, it'll make your life easier.

As for picking out the right clones, I use colony PCR. Forward primer in the vector and a reverse primer in the gene of interest. Pick like 5-10 colonies, spot and number them, and use whatever is left on the tip for your PCR reaction. If you design your primers right, the only positives you get will have your insert in the correct orientation.

The advantages of this over miniprep plus digest and sequencing is that it's wonderful for difficult ligations where you expect a small percentage of colonies containing your insert. I'll go with PCR over miniprep anytime.

 

[lwong]

Why should the incubation be at 4 degrees instead of 14 as indicated in most protocols?

With blunt-end or otherwise nondirectional cloning, you transform and pick up colonies as usual, but when you do a check restriction enzyme digest, you make a strategy which utilizees some restriction enzyme site within your foreign gene and one outside of your foreign gene to determine directionality.

An example:

--|--|-----|-------------------|-----
EcoRI SmaI XhoI SmaI

VERSUS

--|--|--------------------|----|-----
EcoRI SmaI XhoI SmaI

Your gene is cloned into SmaI. Lets say that if the orientation of your clone is correct, XhoI is at the 5' end of your insert. Therefore if you cut with EcoRI outside of the foreign gene and XhoI inside your gene, inserts in the correct orientation will be shorter than the ones in the wrong orientation. Having no insert at all would be obvious, and having multiple inserts may indicate multiple blunt end foreign genes being ligated.

Blunt end ligations are less efficient in general than sticky end ligations, and I suggest that you have a 3-5 to 1 ratio of insert to plasmid when ligating.

Final note: make sure you ligate at 4 degrees, rather than 14 degrees overnight.

I recommend either blue book (Molecular Cloning) or red book (Current Protocols).

 

[huseyin]

Why should the incubation be at 4 degrees instead of 14 as indicated in most protocols?

T4 DNA ligase is a double edged sword in terms of incubation temperature. As you increase the temperature around 22 C, the ligase activity is maximum. However, as you increase the temperature, you are decreasing the probability of insert and cut vector aigning in the right direction.

Therefore, for sticky ends ligating at RT for couple of hours is plenty. However, for blunt ends you have to decrease the temperature so that vector+insert mix is more stable and ligase can do its job.

I do not know how you areated blunt ends:Are you just trying to clone a PCR product?

And, if you use Phosphotase, make sure that you deactivate afterwards(either heat inactivate or coloumn purify it), because in the past I have had some problems because of the residual phosphotase in the ligation reaction.

regards,
huseyin

 

[b&ierstiefel]

Ligase is also less stable at higher temperatures, even though the activity is much higher at these temps. In fact, lifetime of ligase is dramatically lower (we're talking about minutes) at 22 vs. 4 degrees (we're talking about hours).

 

[Doctor&Geek]

Yeah, ditto 😀

 

[lwong]

I'm using blund-end cloning because the site for the enzyme, Hind III, that I used to cut out my gene is not near the promoter on the multiply cloning site in the vector plasmid. Therefore, I can't do sticky-end ligation, but blunt-end.

I do not know how you areated blunt ends:Are you just trying to clone a PCR product?

 

[lwong]

Before selecting which enzymes I'm going to use for the blunt end cloning, I would like to make sure that the enzymes I pick are also not on the plasmid that I'm inserting the gene into. I ordered my plasmid through ATCC, but I can't find a map or sequence to find this information on their site, nor pubmed. Any suggestions to where I can find this info.

Also another question, does anyone have a protocol, or know where I can find a protocol for using T4 polymerase in filling overhangs? I mainly need to know the amount of enzyme I need to add per whatever amount of DNA.

Thanks so much to yall who been so kind and helpful.

 

[Doctor&Geek]

Would help if we knew the ATCC clone you purchased or the plasmid name.

T4 polymerase will eat up your 3' overhangs, so be careful.

xuL, DNA
2uL, 10x T4 polymerase buffer [comes with enzyme]
1uL, 2mM dNTP
H20 to 20uL total reaction mix

+ 1uL [2.5 units usually], T4 DNA polymerase for up to 1ug DNA. Usually amount is not important unless you have >>1ug DNA.

Incubate 5 minutes, 37 degrees.

+ 1uL, 0.5M EDTA to stop reaction.

Phenol/Chloroform extract, ethanol precipitate, perform ligation.

PS:

Even better: http://www.promega.com/tbs/9pim421/9pim421.pdf

 

[lwong]

I won't be back in my lab until tomorrow, but I have two kinds of plasmid vector: they yeast plasmids with AmpR and Ura3 and one has Cup1 promoter and the other has GDP promoter on it. I had found the product onlines since I had the order #, but I couldn't any sequence or map for the plasmid. If I can't access this info directly from ATCC, where could I find a sequence for a commonly used yeast plasmid without too many foreign insertions?

Thanks for the link for T4. I ended up finding some info specific to the T4 from the NEB catalog, which is where I ordered the enzymes from. However, what do you mean that the polymerase will eat up the 3' overhangs? How can I prevent this from occuring?

Thanks again for all your guidance. 🙂

Would help if we knew the ATCC clone you purchased or the plasmid name.

T4 polymerase will eat up your 3' overhangs, so be careful.

xuL, DNA
2uL, 10x T4 polymerase buffer [comes with enzyme]
1uL, 2mM dNTP
H20 to 20uL total reaction mix

+ 1uL [2.5 units usually], T4 DNA polymerase for up to 1ug DNA. Usually amount is not important unless you have >>1ug DNA.

Incubate 5 minutes, 37 degrees.

+ 1uL, 0.5M EDTA to stop reaction.

Phenol/Chloroform extract, ethanol precipitate, perform ligation.

PS:

Even better: http://www.promega.com/tbs/9pim421/9pim421.pdf

 

[Doctor&Geek]

I won't be back in my lab until tomorrow, but I have two kinds of plasmid vector: they yeast plasmids with AmpR and Ura3 and one has Cup1 promoter and the other has GDP promoter on it. I had found the product onlines since I had the order #, but I couldn't any sequence or map for the plasmid. If I can't access this info directly from ATCC, where could I find a sequence for a commonly used yeast plasmid without too many foreign insertions?

Plasmid maps are usually provided by the manufacturer or the original lab that produced it (ancient in most cases for the latter).

Thanks for the link for T4. I ended up finding some info specific to the T4 from the NEB catalog, which is where I ordered the enzymes from. However, what do you mean that the polymerase will eat up the 3' overhangs? How can I prevent this from occuring?

T4 polymerase has 3'->5' exonuclease activity as well (see the above protocol I linked to) which may eat up any 3' overhangs. Use Klenow exo- to avoid 3'--5' exonuclease activity.

Still in the lab -

 

[Marathoner00]

I don't know if you gotten your ligation to work yet. If not, try looking up sticky-end PCR cloning. You run 2 PCR reactions for your insert; each off-set by the sticky end. This way, you don't have to digest your insert (this is where most ligations fail) and you can use non-unique restriction sites.

Another tip for blunt-end cloning. Don't use your own prep of competent cells for transformation. Buy the "library-grade" commercial stuff. It removes one potential item for troubleshooting.

 

[moro]

Hello,

I used NEB T4 DNA pol to make my digested fragment blunt-ended for a ligation but I made a mistake & I forgot the reaction heated inactivating for 40 min instead of 20 min. I was wondering if I should continue this cloning process regardless of this mistake or should I just repeat the digestion & blunt end reaction ?

 

[evo]

Hello,

I used NEB T4 DNA pol to make my digested fragment blunt-ended for a ligation but I made a mistake & I forgot the reaction heated inactivating for 40 min instead of 20 min. I was wondering if I should continue this cloning process regardless of this mistake or should I just repeat the digestion & blunt end reaction ?

Don't think 20 minutes would hurt. I say continue since you are almost done.