Re: [ccp4bb] regarding cloning
HI Vijay, One thing I would like to mention first is that often when a DNA fragment is smaller than or close to 1Kb, it could enter the low EtBr region on an agarose gel if allowed to run to long. Its bound EtBr could be stripped off so significantly that the corresponding band become very faint. Sometimes these could give you false negatives. I would run the gel with accordingly shorter time when expecting small fragments. Or I will chose another pair of enzyme, one of which cuting inside the insert and the other in the vector part. And I will try to choose a pair that give relatively easy to read gel patterns. Or I will try screening the constructs with PCR, which has some other disadvantages as well as some advantages... Another suggestion is to check your insert and vector fragments' concentration by running them on an agarose gel. For fragments longer than 100bp, the brightness of the band should reflect the DNA's total mass - not molarity. The relative molarity is inversely related to the fragment's length. For example, if your insert preparation looks almost as bright as your vector, and the insert is 1.2Kb, the vector is 3.6Kb long, then if you mix them 1:1, you get a 3:1 molarity ratio for the insert:vector. UV quatitation is not very useful for restrictive fragments, because the gel-purified DNA samples are often too diluted. If you do a 300nm~200nm UV spectrum scan for a gel-purified restriction fragment sample, Even without further dilution, you might only see at 260nm there is a very tiny bump sitting on the shoulder of the huge salt peak extends to as far as 290nm - or nothing but the shoulder of the salt peak. But you could still be able to see the DNA on the gel. By the way, I would like to take this chance give a little criticism to the traditional 260~280 two-point reading technique for quatitation of DNA. Although it is kind of a criterion for how complete the protein contaminants have been removed from the DNA preparation (but bear in mind that most proteins have much lower extinction coefficients at 280nm (A=1~2 at 1ug/uL) than DNA at 260nm (A=20 for 1ug/uL), which means this ratio would not be very sensitive to protein contaminants), I regard it as an extremely poor technique for quantitating diluted DNA samples. Since we are now only taking two points, we have no idea if the two points are really representing a DNA peak, or just two numbers taken from the huge salt shoulder that often appear in gel-extraction samples. You may still get a good 260:280=1.8 reading, but what you are actually measuring might be the salt shoulder or 80% coming from the salt. In the early days, UV specs used to have no computer connection, but an electric gauge to give the single point reading, that is why people had to only take a few points - otherwise to assay a plasmid could take a day. There is really no reason to continue doing that these days with computer-controlled UV specs capable of producing UV spectrums at 1nm wavelenth resolution within 5 seconds. To convince myself, I have experimentally proved that the scan and the DNA/RNA program(which does the 2-point) on our lab's Cary50 instrument produce same readings at 260 and 280nm, when properly zeroed (or baselined for the scan program). But with scan you can see the peak and you can substract the salt shoulder. With DNA/RNA, you can fool yourself to be happy with a very poor DNA preparation and use an inflated concentration of that sample. OK, where am I... As to the cases of getting self-ligated vectors, our lab do the following to deal with it: 1. As Raji had already mentioned, use a phosphatase to dephosphorylate the vector. I prefer the Antarctic phosphatase than CIP, for it has lower activity and can be heat-inactivated (and also require Zn2+ cofactor, which is normally not present in ligation mixture - safer). Residual phosphatase activity in you ligation mixture could cause failure of ligation, and CIP is a really strong and robust enzyme. (You might think gel-purification should have removed all the CIP, but, who knows where the CIP runs on an agarose gel? It is just like a native protein eletrophresis. The CIP band can be overlapping your vector band - just a bad possibility.) Whenever I can be sure about the completeness of the double-digestion of the vector fragments, I would avoid using phosphatases, because they could also cause you to have zero colonies on the plate, which is even more distressing. But it is a good method when it works properly, and definitely is among the first things to try when you have self-ligation problems.. 2. If we already have a construct with the same vector and has the same restriction sites, and the insert is relatively long(~1kb or more), I would use that as the vector plasmid for making the vector fragments. When the double digest is working, you can easily see the released insert on the gel when you are doing gel-purification.
Re: [ccp4bb] regarding cloning
Since InFusion now got mentioned, I cant resist, but give my naive non-
expert overview for cloning from the experience in my lab:
1. Typical sub-cloning: Add restriction sites in your primers, make
sure you have a few additional 5' bases overhang else the enzymes will
not cut, digest then your PCR product and put it in your expression
vector. Simply works if you do it nicely, needs restriction enzymes,
make sure these do not cut your gene of interest, ligase, etc.
2. Go through (TOPO)-TA cloning: the main advantage is that ligation
especially in TOPO vectors works really well, you do not need to add
to your oligos the overhangs but just the restriction sites, but then
you need to mini-prep the first ('transfer')vector, cut out your
insert again with enzymes, and put it in your expression vector. No
expression-ready vectors for TA cloning I am afraid, plus you need to
buy the vectors and they are not too cheap for a lot of cloning. In
general, I had found it easier to use, since many times I would not
get colonies when restriction cloning directly to the expression
vector, and it was my personal favorite for a while ...
3. Gateway cloning: Based on specific vectors and enzymes and a
recombination reaction. Although it was big hype, and its certainly a
big success in many areas, in Structural Biology it never delivered,
in my view. The main reason is that vectors were somehow not great for
expression and very often you would get unwanted sequences etc etc.
Need to buy vectors, enzymes, not cheapest.
4. LIC, Ligation independent cloning. It requires that you modify your
favorite vector to be compatible. No need for restriction digest or
ligation (thus it is as cheap as it gets!), no need to design every
oligo differently or think of restrictions sites, no special cells,
just needs a clean PCR product and you can have next morning the
expression cells ready. We use it and like it in my lab and many labs
I know, including quite a few SG projects. A protocol is available
from the CCP4 Community wiki:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/LIC_cloning
If you wish to try it, a His-3C and a no-tag vector for E.coli
expression can be made available from Vangelis Christodoulou,
e.christodoulou.AT.nki.nl , who created these specific vectors and
established the protocol for use in my lab. We have made over the
years about 1,000 constructs many of which yield soluble protein.
Other vectors are also available commercially or from academic
developers.
5. SLIC, Sequence and Ligation independent Cloning. The trick here is
that you do not need to modify your vector first like in LIC, you can
do SLIC in any vector. You need slightly longer oligos than LIC, but
thats fine. People that use it seem to like it, although there are
reports that it can be painful to set up, I hear a lot of positive
things. A protocol is also available from the CCP4 Community wiki
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/The_Milan_protocol_
...
which was contributed my Marina Mapelli, but you need to read a bit
more for how to design the long overhangs since
that issue is not covered in this protocol. To my understanding In-
fusion cloning is basically SLIC.
If LIC was not working well in our lab and we would not had had
vectors we like, SLIC could seem like the way to go.
Apologies if the list is incomplete, and I can admit clear bias
towards my experience. Its only intended on the hope it can be
genuinely useful.
BTW, we (well, Wijnand Mooij in my lab) has developed a very basic
tool to design your cloning oligos for whichever method. The
innovative thing in it (although I am sure there are other tools like
this) is that you input the DNA sequence for your protein, then it
translates it and interrogates a handful of Secondary Structure
Prediction services, Disorder Prediction Services, domain linker id
servers, domain assignment servers, coiled coil servers, and displays
a simple output below your protein sequence. You can then click at the
domain borders you prefer based on this 'condensed' analysis view, to
select possible N- and C-termini for deletion constructs, and click
one button to display all the possible constructs that can be made
with these termini, and the oligos that you need to order for PCR. You
can add to these oligos your favorite overhangs (the default ones are
for one of our His-3C-LIC vector). You are welcome to use it at:
http://xtal.nki.nl/ccd/
ccd: Crystallographic Construct Design
and provide any comments or input. Apologies but there is no help or
documentation, but hopefully its dead simple to use.
Tassos
On Sep 1, 2008, at 16:44, Brian Wengerter wrote:
Alternatively, you could skip troubleshooting digestion/ligation/
etc. and use a kit based on site-specific recombination, like the In-
Fusion kit that Clontech sells. (I don't have any
Re: [ccp4bb] regarding cloning
Hi Vijay, I have heard of TOPO-TA cloning. Not sure what T/A cloning is. I have a couple to check based on your description: 1) Do you CIP-treat your vector? If not, that might be a step to add. Also, you could include a 'vector only' transformation control to determine how many colonies are obtained in the 'vector+insert' plate above background levels. 2) For NheI/BamHI, a sequential digestion (rather than a double digestion) is recommended. 3) Make sure the primers have sufficient extensions (6-8nt) outside of the RE site. 4) Make sure both enzymes work by doing single digestion controls with the vector. It's obviously hard to tell with the PCRT product 4) Titrate vector:insert ratios -- lower and higher than you mention 5)... Once ALL possibilities are exhausted and when nothing else works, I have seen people reorder the exact same primers and then things have worked like a charm! Hope that helps. Raji -Included Message-- Date: 1-sep-2008 03:06:29 -0400 From: vijay srivastava [EMAIL PROTECTED] Reply-To: [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.#194; The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful#194; in subcloning (T/A vector) and getting my insert at 1.2kb after#194; double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how#194; when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/ -End of Included Message--
Re: [ccp4bb] regarding cloning
Hi, First of all - I am curious why did you decide to put in an extra step (the T/A cloning into an intermediate vector)? You can happily digest your PCR product with NheI/BamHI, clean up and ligate into the appropriately digested pET-23a(+). If you have issues, you should definitely try this. Now, since you do have an intermediate step - did you verify that everything was OK after havig subcloned your insert into whatever vector you're using? Did you sequence the insert and most importantly did the sequencing confirm the nature of the linker regions? The enzyme pair that you chose has a slight issue with digestion buffer - most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where Bam still has '100% activity' - however, in buffer 2 you can have star activity of the Bam due to the somewhat lower salt concentration (50 mM instead of the optimum 100 mM). It's not impossible to imagine that you have issues with digestion. This can be easily avoided by sequential digestion although of course it's slightly more work (but if you cut out the T/A cloning step that's actually still faster). So, in conclusion the most likely issue is digesiton (probably of the pET vector, to be more specific). Next likely issue could be ligation - make sure that you base your ligation ratio on the gel intensity of the bands as well as on the OD260 of your DNA. Faulty primers are not likely to be an issue since you seem to be able to restrict your insert out of the intermediate vector. Please note that you can often use SpeI or XbaI instead of Nhe since they have compatible sticky ends. Clearly this depends on the vector you're working with and I am too lazy to look up pET23 polylinker. Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of vijay srivastava Sent: Monday, September 01, 2008 3:06 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. _ Be the first one to try the new Messenger 9 Beta! Click http://in.rd.yahoo.com/tagline_messenger_7/*http:/in.messenger.yahoo.com/wi n/ here.
Re: [ccp4bb] regarding cloning
T/A cloning utilizes the overhangs left by certain polymerases as cloning handles. To lower background, the vectors for TA cloning are often designed to contain a rare cutter which is used during ligation to constantly re-cut the self-ligated vector, so this way only the insert ligation product is supposed to survive. A. -Original Message- I have heard of TOPO-TA cloning. Not sure what T/A cloning is.
Re: [ccp4bb] regarding cloning
I haven't done TA cloning in a very long time. Typically, TA cloning is done with blunt-ended DNA fragments that have been modified by terminal transferase. Normally, a blunt-ended vector digest is treated with terminal transferase and TTP. (If the vector is cut by non-blunt-ended restriction endonucleases, it must be filled in with Klenow or equivalent before adding the T. PCR fragments must be treated with terminal transferase and ATP, or if using Taq (not recommended these days with high-fidelity polymerases available) a significant fraction of PCR products will already have the A overhang. Ligation will result in the insert being inserted (supposedly) randomly in forward and reverse orientations. We don't do this method anymore because it is too problematic and too time-consuming. A double digest with two different overhangs (both vector and PCR product) is much preferred, and results in a high percentage of recombinants with the correct orientation. We usually overdigest PCR products and vectors for at least 3 hr to ensure double-cutting. Phosphatase treatment of the vector after double digest prevents re-ligation of empty plasmid that has only been cleaved by one enzyme. RE digestion of PCR products requires at least 3 additional nucleotides in front of the recognition site. We usually add TGC in front of the recognition site for all PCR primers. Unfortunately in your case, NheI and BamHI have the same 3' C overhang, so double digestion with these two enzymes will result in a random orientation of your PCR product in the vector. I would recommend using a different RE for one of the sites to ensure correct orientation of the PCR product when ligated. We routinely use NdeI and PstI for most of our expression plasmid constructs (assuming these recognition sites are not present in our vector outside the cloning region--this is typically a good pair for pET vectors, and is a good double digest), as NdeI has a start codon within it. (NcoI is another possibility, if your first codon after the start begins with G). For details, see our lab wiki with time-tested methods: http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Protein+Engineering+Protocols Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED] Raji Edayathumangalam wrote: Hi Vijay, I have heard of TOPO-TA cloning. Not sure what T/A cloning is. I have a couple to check based on your description: 1) Do you CIP-treat your vector? If not, that might be a step to add. Also, you could include a 'vector only' transformation control to determine how many colonies are obtained in the 'vector+insert' plate above background levels. 2) For NheI/BamHI, a sequential digestion (rather than a double digestion) is recommended. 3) Make sure the primers have sufficient extensions (6-8nt) outside of the RE site. 4) Make sure both enzymes work by doing single digestion controls with the vector. It's obviously hard to tell with the PCRT product 4) Titrate vector:insert ratios -- lower and higher than you mention 5)... Once ALL possibilities are exhausted and when nothing else works, I have seen people reorder the exact same primers and then things have worked like a charm! Hope that helps. Raji -Included Message-- Date: 1-sep-2008 03:06:29 -0400 From: vijay srivastava [EMAIL PROTECTED] Reply-To: [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.#194; The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful#194; in subcloning (T/A vector) and getting my insert at 1.2kb after#194; double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how#194; when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/ -End of Included Message--
Re: [ccp4bb] regarding cloning
Alternatively, you could skip troubleshooting digestion/ligation/etc. and use a kit based on site-specific recombination, like the In-Fusion kit that Clontech sells. (I don't have any financial interest here--I'm just a graduate student, but I've had good results using it.) The kit is not without its own downsides--it's a bit pricier than traditional cloning, you'd have to design another set of specific primers, and you have to be very careful about the vector:insert ratios you use. Good luck, Brian Artem Evdokimov wrote: Hi, First of all -- I am curious why did you decide to put in an extra step (the T/A cloning into an intermediate vector)? You can happily digest your PCR product with NheI/BamHI, clean up and ligate into the appropriately digested pET-23a(+). If you have issues, you should definitely try this. Now, since you do have an intermediate step -- did you verify that everything was OK after havig subcloned your insert into whatever vector you're using? Did you sequence the insert and most importantly did the sequencing confirm the nature of the linker regions? The enzyme pair that you chose has a slight issue with digestion buffer -- most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where Bam still has '100% activity' -- however, in buffer 2 you can have star activity of the Bam due to the somewhat lower salt concentration (50 mM instead of the optimum 100 mM). It's not impossible to imagine that you have issues with digestion. This can be easily avoided by sequential digestion although of course it's slightly more work (but if you cut out the T/A cloning step that's actually still faster). So, in conclusion the most likely issue is digesiton (probably of the pET vector, to be more specific). Next likely issue could be ligation -- make sure that you base your ligation ratio on the gel intensity of the bands as well as on the OD260 of your DNA. Faulty primers are not likely to be an issue since you seem to be able to restrict your insert out of the intermediate vector. Please note that you can often use SpeI or XbaI instead of Nhe since they have compatible sticky ends. Clearly this depends on the vector you're working with and I am too lazy to look up pET23 polylinker. Artem *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *vijay srivastava *Sent:* Monday, September 01, 2008 3:06 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Be the first one to try the new Messenger 9 Beta! Click here. http://in.rd.yahoo.com/tagline_messenger_7/*http:/in.messenger.yahoo.com/win/
Re: [ccp4bb] regarding cloning
2008/9/1 Artem Evdokimov [EMAIL PROTECTED] Hi, First of all – I am curious why did you decide to put in an extra step (the T/A cloning into an intermediate vector)? You can happily digest your PCR product with NheI/BamHI, clean up and ligate into the appropriately digested pET-23a(+). If you have issues, you should definitely try this. Hello, Artem is right in making this point of course, but why? I think the number of moles of DNA molecule that you get is important. With PCR you can get a huge number of moles, especially for a very intense 600bp band. As you go up in size of product, the yield often becomes lower, and it becomes more difficult to clone, but still, you'll probably have more moles of insert compared to using restriction enzymes to cleave a fragment out of a vector. So when is using a TA cloning vector step justifiable? If you have a meagre PCR product which you really can't clone any other way, TA cloning is the most efficient with these low yields (for me). Can high PCR yields inhibit restriction enzyme-based ligations? Yes, if you don't cleave these products to completion. If you have PCR products where a significant proportion are not cleaved properly, the non-cleaved molecules will have an inhibitory effect on your reaction- one can imagine that they'll mop up a large proportion of your vector and make it unusable. So, if you have a huge PCR product, try cleaving 10-30% of the entire reaction either 4 hours or over night to make sure it's fully cleaved. (Again, as Artem says, choose your buffer carefully here). As a final piece of advice, make sure you have a copy of Molecular Cloning, a Laboratory Guide (Cold Sring Harbor Press) nearby. http://books.google.fr/books?id=YTxKwWUiBeUCdq=molecular+cloning+a+lab+manualpg=PP1ots=FVO87K4uEtsig=xTYcKpFP45dBqwsfRWZ0UXR0wb8hl=ensa=Xoi=book_resultresnum=1ct=result ...You will probably have a recent or not-so-recent edition (A.K.A. Maniatis for previous editions IIRC) in a University Library nearby. I recently frog-marched a technician here to read it when he was having cloning problems. After persuading him that the PEG-additive protocol might be helpful to help him with a particularly nasty cloning problem, he tried it. As he said, the results were miraculous. (I have no affiliation to CSHL press nor the authors BTW) Good luck, Mark Now, since you do have an intermediate step – did you verify that everything was OK after havig subcloned your insert into whatever vector you're using? Did you sequence the insert and most importantly did the sequencing confirm the nature of the linker regions? The enzyme pair that you chose has a slight issue with digestion buffer – most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where Bam still has '100% activity' – however, in buffer 2 you can have star activity of the Bam due to the somewhat lower salt concentration (50 mM instead of the optimum 100 mM). It's not impossible to imagine that you have issues with digestion. This can be easily avoided by sequential digestion although of course it's slightly more work (but if you cut out the T/A cloning step that's actually still faster). So, in conclusion the most likely issue is digesiton (probably of the pET vector, to be more specific). Next likely issue could be ligation – make sure that you base your ligation ratio on the gel intensity of the bands as well as on the OD260 of your DNA. Faulty primers are not likely to be an issue since you seem to be able to restrict your insert out of the intermediate vector. Please note that you can often use SpeI or XbaI instead of Nhe since they have compatible sticky ends. Clearly this depends on the vector you're working with and I am too lazy to look up pET23 polylinker. Artem -- *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *vijay srivastava *Sent:* Monday, September 01, 2008 3:06 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. -- Be the first one to try the new Messenger 9 Beta! Click here.http://in.rd.yahoo.com/tagline_messenger_7/*http:/in.messenger.yahoo.com/win/ -- Mark BROOKS Telephone: 0169157968 Fax: 0169853715 Institut de Biochmie et de Biophysique Moleculaire et Cellulaire
