molecular cloning

[Och]

Hi all,

 

Please I'm having some very serious problems on my project. What I am trying to do is to:

1. extract an EGFP gene from pSAT1-EGFP-C1 (4553 bp) and clone it onto the binary vector, pPZP-RCS2-ocs-bar-R1 (8637 bp)and then transform it with competent cells.

2. Later on, I will use it to clone some genes from tomato plants by silencing usin VIGS.

 

But the problem I am having now is in the first step. I havn't been able to clone the EGFP gene onto the binary vector. The protocol I have been using is:

For the vector

(a) plasmid extraction of vector (plasmid conc. is usually high)

(b) enzyme digestion of vector with Asc1 enzyme (I get one band after cutting)

© agarose gel purification of vector

(d) measurement of vector DNA concentration using nanodrop (sometimes I get low conc.)

 

For the insert

(a) plasmid extraction of insert (plasmid conc. is usually high)

(b) enzyme digestion of insert also with Asc1 enzyme (I get 2 bands after cutting, then I use the lower band, with size of like 1600bp, for gel purification)

© agarose gel purification of insert

(d) measurement of insert DNA conc. (also I get very low conc. of DNA)

 

Then I go ahead with ligation of the vector and insert by adding 4ul of insert to 1ul or 2ul of vector; 1ul of T4 DNA ligase; 1ul of T4 DNA ligase buffer; and add up with ddH2O in a 10ul reaction and incubate @ 16oC overnight.

 

After that, I then go ahead with transformation using competent cells that were ordered and tested. I grow the ligation mix on spectinomycin selective media since the vector has spectinomycin resistance for 16 hours. But at the end, I will either: not see any growth; or see some king of very tiny colonies that won't grow on LB media.

 

Please, I really need the help of this forum to know what the prob is. I have been stuck here for several months now. I will really appreciate all kinds of opinions on this.

[CharonY]

What is the concentration ratio of the of the insert to vector? However, seeing now resistant bacteria (regardless of insert) tells me that either that there are problems during gel extraction, ligation, or transformation. The reason being that even with no insert, the plasmid should re-ligate yielding some resistant colonies.

 

Since you mentioned that you tested the transformation (with the same plasmid?) it appears that the ligation or the gel extraction may be at fault. After extraction from agarose you may want to run a test gel whether you actually got plasmid isolated (also avoid putting it on UV for too long). My suggestion is to make a series of controls, e.g. re-ligate the vector without agarose purification (with and without adding insert) and transform it, cut and re-ligate the vector alone and transform the cut and the re-ligated vector (to check the ligation rate) and possibly test transformation.

 

My guess is that the agarose purification may not have worked out well, but without controls it is really hard to tell.

[Och]

@ CharonY. Thanks a lot for your invaluable reply. I will do some controls.

I did not test the transformation. What I meant was that I tested the competent cells to make sure that they're okay.

What do you mean by the concentration ratio of the of the insert to vector? I've not been able to figure out how to calculate the conc. ratio of the insert to vector. I will appreciate if you can help me with that.

 

Please what do you mean by 're-ligate the vector without agarose purification '? Do you mean that I should ligate the vector after plasmid extraction and enzyme digestion?

 

Thanks.

[CharonY]

Essentially you calculate how much vector and insert you have (based on conc measurement and adjustment by bp). I.e. you just multiply the ng of whatever DNA you have with the bp length. It is not absolutely precise, but often close enough. Usually a slightly higher amount of insert is used (around 3:1). Though in the end it is quite a bit trial and error.

 

Please what do you mean by 're-ligate the vector without agarose purification '? Do you mean that I should ligate the vector after plasmid extraction and enzyme digestion?

Precisely. If you do tha you can figure out how efficient your ligation is (by transforming the digest and by transforming the ligated plasmid and comparing the number of transformants).

 

But as the first step I would now suggest checking whether your cells are still competent (i.e. transforming your isolated plasmid).

[Och]

@ CharonY. If for example I have 20ng vector DNA with 8000bp and 10ng insert with1600bp; so i will say 20*8000: 10*1600? how will I know the volumes of each to add to the ligation mix?

 

So if I transform the digest(do you mean without ligation), should I see colonies?

 

per the first step, do you mean transforming the plasmid after extraction from bacterial liquid culture?

 

what is the reason for re-ligating the vector without agarose purification (with and without adding insert) and transforming it?

 

Thanks a lot. I really appreciate your response.

[CharonY]

If you used the nanodrop you should get a concentration. Thus you know how many ng you got per ml, for instance. Based on that you can e.g. start with one microliter of insert and calculated how much you need to have 1:3 molar ratio (generally low volumes are used so that dilutions with the ligation buffer are easier).

 

For the next questions in order:

- You should see few colonies if the digest worked. There are almost always a few plasmids that do not get restricted (you won't see them on the gel, usually). This is just to control for restriction and to be able to calculate the ligation efficiency.

 

- Yes, this is just to test that your vector is properly transformed with your protocol.

 

- This is to test whether you got some issues with the extraction from gel. And if you compare the amount of colonies from a simply re-ligated vector to the digest (if you transform with similar concentrations) you can see whether your ligation was efficient. If e.g. the number of colonies is the same as the digested vector, it would indicate that the ligation did not work. If everything works except your approach after agarose purification it may indicate an error at that step.

[Och]

Thanks CharonY. When I want to ligate the unpurified vector, should I purify the insert or should I also leave it unpurified? That is, should I ligate the vector and the insert without purifying any of them?

 

Also, do you think I can get the gene cloned onto the vector if I purify the vector(spectinomycin resistance) and do not purify the insert (ampicillin resistance)?

 

 

Thanks.

[CharonY]

Remind me, the insert was cut out from an existing vector? If so you can also try to ligate without further treatment. I do not understand the last sentence. Does the insert encode amp resistance, or the vector from which it is derived?

[Och]

the vector from which the insert is derived is encoded with amp resistance.

 

Yes, the insert was cut out from an existing vector.

 

Per further treatment, do you mean gel purification?

[Mark Ian]

this helped me allot.

 

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cloning_troubleshooting_guide.asp#.UDJUnXU0WSo

 

If you're using electroporation be sure to keep everything on ice, and quickly getting you're bacteria on medium (they literally are in shock). Remember that every second you take will reduce the competence of your bacteria, so work fast.

 

I prefer having two samples, with different ratios of vector to insert (1:3 and 1:7), it may be that the samples with higher concentration will explode, but if it works it usually yields more colonies. Also, since your insert isn't too long you can try amplifying it out instead of the gel extraction. Gel extraction can result in low yield of insert. Sometimes the bacteria simply do not grow well, and you have to work with what you get. I would just pick a few colonies and then digest with your enzyme to see if the insert is there or not.

Edited August 20, 2012 by Mark Ian

[mamajohns]

normally you should use two enzymes in restriction digestion, and after cutting you should see two bands in your gel, or three by a incomplete digestion. Otherwise you use T4 ligase as a middle reaction.

 

 

 

 

http://www.assay-protocol.com