Problem with cloning - what is wrong? - (Jan/04/2013 )

Hello everybody,

I´m new here and need you advice. This is my problem:

I want to clone specific sequence into the specific vector position. The position is the HindIII restriction site. I created my own specific sequence and let it synthetized, This sequence has 170 bp in lenght. After that I designed primers, which introduce HindIII restriction sites to the ends of 170 bp sequence.

Here is the beginning of forward primer: 5´-GAGAAAGCTTC.... -3´ (HindIII restriction site is in bolt).

Here is the beginning of reverse primer: 5´- TAGGAAGCTTG... -3´ (HindIII restriction site is in bolt).

After optimization of PCR I obtained amplicons which should contain HindIII restriction sites at the ends. Purification of PCR product was performed with QIAquick PCR Purification Kit according to manufacturer guideline. Than I measured concentration and purity of PCR product on nanodrop: c = 120 ng/microliter and 260/280 nm = 1,83, 260/230 nm = 2,6.

Than i performed overnight restriction digestion with HindIII: 40 microliters of purified PCR product (cca 4800 ng DNA), 31 microliters of water, 8 microliters of buffer B and 1 microliter of HindIII (100,000 U/ml). Purification of digested PCR product was performed next day using again QIAquick PCR Purification Kit, measure on nanodrop gave the following values: c=127 ng/microliter and 260/280 nm = 1,81, 260/230 nm = 1,86. Digestion of PCR product with HindIII was verified by agarose gel electrophoresis, digested PCR product was smaller then undigested.

Than I isolated vector from bacterial culture using Macherey-Nagel NucleoSpin Plasmid Kit according to manufacturer instructions. Than I measured concentration of vector on nanodrop: c = 92 ng/microliter and 260/280 nm = 1,86 and 260/230 nm = 1,99. Than i performed overnight restrition digestion with HindIII: 11 microliters of vector (cca 1000 ng DNA), 6 microliters of water, 2 microliters of buffer B and 1 microliter of HindIII (20,000 U/ml).

Next day I performed dephosphorylation. I added CIAP directly to the restriction mixture. I added 0,5 microliter of CIAP and let the tube stay in room tempetature for 30 minutes and after I added next 0,5 microliter of CIAP and let it stay for another 30 minutes. (I use this CIAP from NEB: http://www.neb.com/nebecomm/products/productm0290.asp ). It is good approach how to dephosphorylate the vector or not?

After dephosporylation i loaded the mixture into the agarose gel (GTG SeaKem) and extracted the digested and dephosphorylated vector with QIAquick Gel Extraction Kit according to manufacturer instructions. Than I measured concentration and purity of vector on nanodrop: c = 9,5 ng/microliter and 260/280 nm = 2,3, 260/230 nm = 0,02. But a nice band was clearly visible on control agarose gel...


According to NEB Quick Ligation Kit (http://www.neb.com/nebecomm/products/productM2200.asp) I prepared ligation mixtures and after that I tried to transform E. coli BL21 (DE3). Than I selected potential tranfromants with kanamycin LB plates (30 micrograms/microliter). But i did not acquire colonies.

So I tried to do another lagation mixtures, but still nothing grows. So i tried this mixture (ratio 1:5, vecrot:insert), insert has 154 bp and vector 7100 bp:

5,2 microliters of vector ( 50 ng DNA)
3 microliters of insert (PCR product, 3,3 ng DNA)
1,86 microliters of water
10 microliters 2x ligation buffer
1 microliter of ligase

and let it ligate in 16 °C overnight

Than I tried to transform E. coli BL21 (DE3) and selected potential tranfromants with kanamycin LB plates (30 micrograms/microliter). After 2 day of cultivation I obtained some big collonies and a lot of small colonies. The small colonies are not resistant against kanamycin when they are tranferred into liquid LB broth with kanamycin. The big colonies are resistant but they do not contain recombinant plasmid :-(

Would it be possible to tell me what is wrong? Thanks. :-)

Well , check your primer again but it is always better to have two different RE . You do not have a direction problem or multimerization of insert. Furthermore, your insert and vector is really big in size, so check again different ligation condition. The big colonies mean that your CIAP reaction was not completly successful.

Transforming into BL21 strains initially is a bad idea, since the transformation efficiency is quite low. You should transform into a cloning strain such as DH10B or DH5a first, then miniprep large amounts of your plasmid, and then transform into BL21.

As Pangea pointed out, it would be much better to have two different RE sites in your vector and insert, which would allow you to clone without desphosphorylation (which often introduces problems) and would allow directional control over the insert.

Thank you for answers. I need to clone my insert into the HindIII place of the vector so I can not use 2 RE:|

I will try to clone to E. coli TOP10F or DH5alpha :-) Thank you for this advice.

Do you have experience with NEB CIAP which I use? How to set up reaction?

I have also doubts about the compositon of ligation mixtutes. Because of small size of insert (154 bp) and huge size of vector (7100 bp) a add 50 ng of vector DNA and only 1 ng of insert DNA and this is 3:1 ratio of vector:insert. To add such a small amouth of insert I have to dilute it and and do not believe that it is correct:| Do you have any experience with cloning such a small inserts to big vectors? Thank you, each advice is good:-)

You will need to deactivate the CIAP before you can use the product for anything further, heat treating for 30 min and 95 degrees C is supposed to work (though doesn't in my experience). You could clean up with a phenol-chloroform step, which will remove the CIAP. If you must dephosphorylate try and use one of the heat sensitive versions, as these can be deactivated by heat.

Follow the instructions on the product sheet for the CIAP as to how to set up the reaction.

How much of the ligation mix are you using to transform - you should get better transformation if the ligation mix makes up 5% or less of the volume of the cells.

According to your recommendations I have found a way of how to clone with two different RE. But I have another question, the restriction enzymes are HindIII and NotI and the distance between the places where they digest in vector is only 7 bp. Both enzymes work in different buffers so I can not digest simultaneously. First I want to digest with NotI, clean the plasmid with QIAquick PCR Purification Kit and then digest with HindIII (there will be 7 bp before HindIII restriction site so the digestion might not be problem).

​Is it necessary to perform dephosphorylation?

How to clean the vector after each digestion? Is it good idea to use QIAquick PCR Purification Kit or it is much more better to use QIAquick Gel Extraction Kit? Thank you for your answers :-)

The double digest tool at NEB suggests buffer 2 + BSA for simultaneous digestion, or, if using the -HF versions, buffer 4 + BSA. In either case, following digestion, you can simply heat kill these enzymes rather than purifying the product. Unless you need to separate out the vector from the insert, you don't need to do a gel separation. You definitely don't need to dephosphorylate. Think always about ways to do what you want to do with the smallest number of steps and fewest purifications.