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Multiple ligation - How many digestion products is it possible to ligate at the same time ? (Nov/20/2006 )

Some restriction enzymes cut outside their recognition site and live unspecific sticky ends (Bgl 1, Bpi 1, Esp3I...). When you want to assemble several fragments at the same time, you can take advantage of this property by designing the cutting sites of your fragments so that their sticky ends will match with one another. (end cutting site of fragment 1 should be the same as begining site of fragment 2, end cutting site of fragment 2 should be the same as begining site of fragment 3 ...)

My question is: How many fragments can you assemble at the same time in this way ??? And what is the best enzyme to do it ??

Thx, David

-dbikard-

I only have experience with 2 fragments, seem not difficult, but not sure the efficiency of ligation more fragments...

-rye-

The largest I have done as part of my regular work is 5 way ligation with usage of colour testing. (4 inserts and 1 vector) The sucess ratio was about 1 in 20 to 1 in 30 colonies. Stocastic effects however is very strong, so it is possible to have 40 negatives in a row followed by 3 correct colonies. A large screen is thus required.

However as I have a multichannel pipette biggrin.gif and screen by colony PCR , I have no problem with such success ratios. Screening 24 colonies takes about as much effort as screening 96 or even 192 colonies.

I believe a 6 way ligation is easily doable.

Enzymes that I use are as follows

Compatibility groups.

Xho / Sal (Sal is only use to cut PCR products)
BglII/BamHI/BclI (BclI is only use to cut PCR products)
XbaI/NheI/SpeI
NotI pfil/ XmaI pfil

Other enzymes used, Not, EcoRI, HindIII, ClaI, SacI, NdeI, Asp718, PstI.

The Xho and BamHI compatibility groups are compatible with each other if they are partially filled in. (pfil)

Aways use IPTG/Xgal colour testing to discrimated between empty vectors and ones with some kind of insert. Always dephosphorylated the vector.

-perneseblue-

QUOTE (perneseblue @ Nov 20 2006, 09:23 PM)
The largest I have done as part of my regular work is 5 way ligation with usage of colour testing. (4 inserts and 1 vector)

Enzymes that I use are as follows
Compatibility groups.
Xho / Sal (Sal is only use to cut PCR products)
BglII/BamHI/BclI (BclI is only use to cut PCR products)
XbaI/NheI/SpeI
NotI pfil/ XmaI pfil

Other enzymes used, Not, EcoRI, HindIII, ClaI, SacI, NdeI, Asp718, PstI.
The Xho and BamHI compatibility groups are compatible with each other if they are partially filled in. (pfil)

Aways use IPTG/Xgal colour testing to discrimated between empty vectors and ones with some kind of insert. Always dephosphorylated the vector.


Hi, probably you are very succesful in cloning smile.gif I have some questions to you:

1. In Enzyme compatibility groups, what do you mean under "is only use to cut PCR products"? Why only PCR and why do you cut PCR products? Probably, to have sticky ended PCR products?
I was told: since the introduced restrictase sites are very close to the 5' and 3' ends in PCR products (we add just "AA" after the sites in the primer), the restrictases do not cut them efficiently and mostly you do not have sticky ends there.
Therefore we clone PCR products directly (blunt ended), let's say into EcoRV cut pBluescript SK+, so that I can use the color selection. Then I sequence the plasmid. When I have a correct insert, I subclone it to the target vector.
Do you have some efficient protocol of digestion to introduce sticky ends into PCR products

2. The use of compatibility groups is very efficient, I see. What software do you use to plan clonings? OR where can I find the list of compatibility groups to plan the cloning efficiently?

Sincerely, Proteos

-Proteos-

QUOTE (Proteos @ Nov 21 2006, 06:23 PM)
Hi, probably you are very succesful in cloning smile.gif I have some questions to you:

1. In Enzyme compatibility groups, what do you mean under "is only use to cut PCR products"? Why only PCR and why do you cut PCR products? Probably, to have sticky ended PCR products?
I was told: since the introduced restrictase sites are very close to the 5' and 3' ends in PCR products (we add just "AA" after the sites in the primer), the restrictases do not cut them efficiently and mostly you do not have sticky ends there.
Therefore we clone PCR products directly (blunt ended), let's say into EcoRV cut pBluescript SK+, so that I can use the color selection. Then I sequence the plasmid. When I have a correct insert, I subclone it to the target vector.
Do you have some efficient protocol of digestion to introduce sticky ends into PCR products


SalI is a rather problematic enzyme. Often but not always, I find plasmids cut with SalI will not ligate efficiently. I have no idea why. PCR products cut with SalI on the otherhand will ligate. Thus, to save myself grief, I will only use SalI to cut PCR products.

BclI is dam methlyated and the cloning strain my lab uses, XL1blue is dam+. Thus any BclI RE site introduce into my XL1blue will become methlyated and will become resistent to the BclI resistriction enzyme. So in my case, BclI can only be used on PCR products.

QUOTE (Proteos @ Nov 21 2006, 06:23 PM)
I was told: since the introduced restrictase sites are very close to the 5' and 3' ends in PCR products (we add just "AA" after the sites in the primer), the restrictases do not cut them efficiently and mostly you do not have sticky ends there.


I hope you added more then 2 As.... and even if you did, A and T isn't a good thing to add to the 5' terminal of a primer.

Look up
NEB restriction enzyme tools
NEB technical reference

And check the cleavage close to end.... You will see the cutting efficiency of the various enzymes close to DNA ends. On average most restriction enzymes need at least 5bp of overhang to work. Some need even large overghang 9bp.

If you design your primers according to what NEB says, then digestion of your PCR products would be like any digest....

PCR...
Then clean PCR product by either a kit or gel purification. It is important to remove the polymerase else it will fill in your overhangs. Also note, themophilic polymerase (that is every polymerase that uses a thermocycler) is resistent to phenol.
Then digest.

As for adding AAs = bad!
The reason being that when a primer binds to the template... the initial binding may not be perfect. The primer may curl the template, missing several base pairs. Furthermore the moment the 3'end anneals with the template, the polymerase starts.. being able to withstand upto 1bp misspairing.

So for more accurate priming, it is ideal to design a primer where there is a gradient of GC content... binding affinity. With riches GC content (and thus strongest binding affinity) on the 5'end and poorest on the 3'end... So the primer can bind relative strongly to the template via the 5'end but leave the 3'end free to scope about looking for perfect alignment.

However designing such a primer is nearly impossible... so the second best would be to make the guard... the 5'end of the primer after the RE site more GC rich....(Just don't make it all GC though)



QUOTE (Proteos @ Nov 21 2006, 06:23 PM)
2. The use of compatibility groups is very efficient, I see. What software do you use to plan clonings? OR where can I find the list of compatibility groups to plan the cloning efficiently?


I use Vector NTI to plan my cloning. The software is free... legally Infomax gives a free 1year licence for VectorNTI.... there are also free programes that do similar things a vector NTI, but I don't remember their names.

As for compatability groups again look up NEB...Compatible Cohensive Overhangs....
Fermentas also does the same.

-perneseblue-

QUOTE (perneseblue @ Nov 24 2006, 01:35 AM)
I hope you added more then 2 As.... and even if you did, A and T isn't a good thing to add to the 5' terminal of a primer.
......
As for adding AAs = bad!
The reason being that when a primer binds to the template... the initial binding may not be perfect. The primer may curl the template, missing several base pairs. Furthermore the moment the 3'end anneals with the template, the polymerase starts.. being able to withstand upto 1bp misspairing.

So for more accurate priming, it is ideal to design a primer where there is a gradient of GC content... binding affinity. With riches GC content (and thus strongest binding affinity) on the 5'end and poorest on the 3'end... So the primer can bind relative strongly to the template via the 5'end but leave the 3'end free to scope about looking for perfect alignment.

However designing such a primer is nearly impossible... so the second best would be to make the guard... the 5'end of the primer after the RE site more GC rich....(Just don't make it all GC though)

I use Vector NTI to plan my cloning.

I understand, so you suggest instead of AA to use for instance 5'GCGCGAA-RE1_Site- and then the complementary part of the gene? Or after the RE1_Site again some additional nucleotides and then the gene? I did not really understand you regarding 3' choices. Conversely, I was told to choose the 3' end (the gene part) so that it ends with GC. Is it a bad choice?
-------
My strategy:
I design primers by simple scheme: 5'-AA-RE1_Site-RE2_Site-Gene. The same is true for the reverse primer.
Thus, I have just two A-s in the beginning, then comes the RE site (maybe the 2-nd RE site as well) and the complementary part of the gene. I do not include any additional nucleotides there...
...And it works for blunt end ligation with, let's say, EcoRV cut pBluescript SK+. I use this vector most of the time for the PCR products since it has a blue-white selection posibility.
-------
I also have Vector NTI I wanna use it but it's huge and too complicated. I do my stuff much more quickly in Clone Manager (although I have very old ver5.02).


Your comments?

-Proteos-

HyTK Gene

gccgccaccatgaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggag
ggcgaagaatccgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttct
acaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaattcagcg
agagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttc
tgcagccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgc
aaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtga
tggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggc
acctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgagg
cgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgc
gctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaac
tctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggag
ccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtcgcgtctgcgt
tcgaccaggctgcgcgttctcgcggccatagcaaccgacgtacggcgttgcgccctcgccggcagcaagaagccacggaag
tccgcccggagcagaaaatgcccacgctactgcgggtttatatagacggtccccacgggatggggaaaaccaccaccacgc
aactgctggtggccctgggttcgcgcgacgatatcgtctacgtacccgagccgatgacttactggcgggtgctgggggctt
ccgagacaatcgcgaacatctacaccacacaacaccgcctcgaccagggtgagatatcggccggggacgcggcggtggtaa
tgacaagcgcccagataacaatgggcatgccttatgccgtgaccgacgccgttctggctcctcatatcgggggggaggctg
ggagctcacatgccccgcccccggccctcaccctcatcttcgaccgccatcccatcgccgccctcctgtgctacccggccg
cgcggtaccttatgggcagcatgaccccccaggccgtgctggcgttcgtggccctcatcccgccgaccttgcccggcacca
acatcgtgcttggggcccttccggaggacagacacatcgaccgcctggccaaacgccagcgccccggcgagcggctggacc
tggctatgctggctgcgattcgccgcgtttacgggctacttgccaatacggtgcggtatctgcagtgcggcgggtcgtggc
gggaggactggggacagctttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggcccacgacccc
atatcggggacacgttatttaccctgtttcgggcccccgagttgctggcccccaacggcgacctgtataacgtgtttgcct
gggccttggacgtcttggccaaacgcctccgttccatgcacgtctttatcctggattacgaccaatcgcccgccggctgcc
gggacgccctgctgcaacttacctccgggatggtccagacccacgtcaccacccccggctccataccgacgatatgcgacc
tggcgcgcacgtttgcccgggaGATGGGGGAGGCTAACTGA




Primer
ORFHyTK-IHind3/BglII-F
HindIII : AAGCTT
BglII : AGATCT
Spacer : ACT
Guard :[ b]CGACTG[/b]

Binds to 5’end of HyTK : GCCGCCACCATGAAAAAGC tm = 60

CGACTG AAGCTT ACT AGATCT GCCGCCACCATGAAAAAGC


ORFHyTK-IBamHI-R
BamHI : GGATCC
3’end of HyTK : GATGGGGGAGGCTAACTGA
Binds to 3’end of HyTK : TCAGTTAGCCTCCCCCATC (Reverse complementary) tm = 60
Guard : CAGTCA

CAGTCA GGATCC TCAGTTAGCCTCCCCCATC

--------------------------------

Hopefully this gets my idea over clearly.

You have to have more then 2bp from either end of the restriction site for digestion to be efficient.

GC clamps are nice...(on the 3'end) but that shouldn't rule the primer location.

I ment by GC gradient... is the section of your primer that binds to the template should ideally have a GC gradient.... from high GC at the 5' to low GC at the 3'. This gradient is independent of the tinny GC clamp at the 3'.

As mentioned before, this GC gradient is usually unattainable in real life. So I make the Guard at the 5'end of the primer GC rich.

Note, that few restriction enzyme will cut efficiently at sites only 2bp from the end. Thus your troubles.

-perneseblue-