Hi Prashant,

in our experience, we always had problems with 3-component ligations. In my opinion, you should go for the whole gene. What were exactly the problems you had? Didn't you get any PCR product or no clones after ligation? In the case of no PCR product, you should try a high performance polymerase (e.g. NEBs Q5, they could send you a trial package), DMSO and/or a gradient for the annealing terperature. Sometimes the optimal annealing temps are way off the calculated ones (mostly below). High GC-content (above 50%) is problematic - DMSO is the cure. In the case of ligation problems, there could be several problems: 1. at the level of restriction digest - consider adding overhangs for the restriction enzymes to your PCR primers (CG before GGATCC/BamHI, a single G before GAATTC/EcoRI and CCG before CTCGAG/XhoI), 2. vary the insert to vector molar ratio in your ligation (1:6, 1:4, 1:2, 1:1. 2:1. 4:1. 6:1) - we usually start with 4:1. 3. Make ligations of 10 µl volume and transform into 90 µl of highly competent E. coli TOP10 or DH5a cells (made by this protocol: http://openwetware.org/wiki/TOP10_chemically_competent_cells). Make colony PCR afterwards (with short primers binding in the T7 promotor and terminator regions, which you can use for all your cloning into pET vectors, as they bind within the vector sequence, max. T anneal 58°C) to quickly check for positive clones. 1./2. and 3. you can also consider, if you want to stick to the 3-component ligations (2 inserts, 1 vector). Good luck, Magdalena

Some questions before this question can be answered.

1. Are you doing the cloning using blunt or sticky end ligation?

2 Which enzymes and ligase are you using?

3. Has your ligation buffer been through many freeze-thaw cycles?

4. What is your bacterial strain and transformation protocol?

5. Why are you doing it in two parts? Is this absolutely necessary?

6. What are the details of the two-part cloning?

7. Can you use a non-ligase method such as Gateway cloning?

8. Does the target DNA have anything expressed that might be toxic to E. coli?

The answers to these will lead to the solution to your problem.

Dear Deepan Shah:

Following are the answers of your question:

1.I am doing sticky end Ligation.

2.i am using BamH1 and Xhol for full gene

for part 1:BamH1 and EcoR1

For part 2:EcoR1 and Xhol

3.I have kept it in aliquotes still i am using it at least for five freeze thaw.

4.DH5 alfa

5.It is not necessary to do it in parts but i was not successful in full gene .It would be much better if i can clone full gene .Will be best for my study.

6.No specific detail same after RE going for Ligation then transformation.

7.I don't have any idea about gateway cloning or will it be OK for expression and further study.

8.No there is nothing toxic in the gene.

As per my experience of the experiments i have answered the above questions still if you feel you need more information plz let me know .If you are having any idea from these answers Could you please help me how can i get the clone.

looking for your positive response

Thanking you

Hi Prashant,

in our experience, we always had problems with 3-component ligations. In my opinion, you should go for the whole gene. What were exactly the problems you had? Didn't you get any PCR product or no clones after ligation? In the case of no PCR product, you should try a high performance polymerase (e.g. NEBs Q5, they could send you a trial package), DMSO and/or a gradient for the annealing terperature. Sometimes the optimal annealing temps are way off the calculated ones (mostly below). High GC-content (above 50%) is problematic - DMSO is the cure. In the case of ligation problems, there could be several problems: 1. at the level of restriction digest - consider adding overhangs for the restriction enzymes to your PCR primers (CG before GGATCC/BamHI, a single G before GAATTC/EcoRI and CCG before CTCGAG/XhoI), 2. vary the insert to vector molar ratio in your ligation (1:6, 1:4, 1:2, 1:1. 2:1. 4:1. 6:1) - we usually start with 4:1. 3. Make ligations of 10 µl volume and transform into 90 µl of highly competent E. coli TOP10 or DH5a cells (made by this protocol: http://openwetware.org/wiki/TOP10_chemically_competent_cells). Make colony PCR afterwards (with short primers binding in the T7 promotor and terminator regions, which you can use for all your cloning into pET vectors, as they bind within the vector sequence, max. T anneal 58°C) to quickly check for positive clones. 1./2. and 3. you can also consider, if you want to stick to the 3-component ligations (2 inserts, 1 vector). Good luck, Magdalena

Thanks for the information Prashant. The combined pET28 and your insert will be close to 10 kb in size.

Cloning large fragments can be problematic but not impossible. The main issues are shearing of the ligated 'open circle DNA' and reduced transformation efficiency so you need to optimise everything. Here are my recommendations:

1. Old stocks of XhoI can sometimes become non-functional and lead to incomplete digestion. Moreover XhoI sites can be blocked by overlapping dcm methylation. So check that you have no methylation. I've never had trouble with EcoRI but some manufacturers recommend specific buffers so ensure that both enzymes cut efficiently in the conditions you are using. Never had issues with BamHI.

2. If you are cloning a PCR fragment ensure that you have at least 4 extra bases at either end before the restriction sites.

3. If you are using gel purification of bands for ligation then minimise UV exposure and do not vortex the DNA at any stage. This is worth repeating: Do Not Vortex the DNA. No vigorous pipetting up and down either. Just flick the tubes gently if you must mix.

4. Most ligation protocols recommend that you use a 3:1 insert to vector molar ratio. However, in my experience this isn't always optimal so set up several ligations with vector and insert at different molar ratios plus a self ligation control. If possible also set up a positive control reaction that you know works.

5. Ensure that the DH5 alpha are of a sufficient competency to give at least 10^8 cfu/ug of supercoiled plasmid more if possible. We make our own competent cells using OD600 0.4 cultures and the RbCl2 method.

6. Do not let the competent cells get warmer than ice so pre-chill your ligations before adding to competent cells.

7. Use less DNA in the transformations (between 5-10 ng total DNA max.). Too much DNA can inhibit transformation efficiency.

8. Incubate the ligation plus competent cells on ice for at least 1h and heat shock at 37C. Try one set at 42C also but in my hands 37C for 90s works better for large DNAs.

9. After heat shock place on ice again for 10 min and then add 0.1 mL of LB or other growth medium and incubate at 37C for at least 1 h to allow the kanamycin resistance gene to express so that the cells containing your clone are resistant when you plate them.

10. Plate the whole of your transformation mix on a plate.

I hope these tips help you to get the full sized clone.

You probably know most of this stuff already!

Keep us informed how it goes.

Good luck

Deepan

Dear Magdalena Schacherl ,and Sezer Okay Thanking you very much for the useful suggestions.

I will get back to you if facing other problms....

Dear Deepan Shah,

Thanking you very much for the information .I will be doing the changes as per your suggestions .Will also be changing molar ratio.As i am using the fresh Xhol may be 5-7 day back i received it shd not hv any prblm.

Will be back if there is still a problm.

Thanking you