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Sub-cloning and transformation


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17 replies to this topic

#1 och

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Posted 11 July 2012 - 08:18 AM

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.

#2 Nayeem991

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Posted 11 July 2012 - 10:11 AM

Hi, I think there is prob in the ligation step. It might be because of the ligase buffer or ligase. Did you check the efficiency of your ligase? You can prepare a reaction mix without the insert and transform that into the competent cell. All the condition should be same, just use ddH2O instead of insert. If you find no colonies yet, i think you should change your ligase and ligase buffer and try it again.

#3 phage434

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Posted 11 July 2012 - 11:52 AM

Start by making sure you can transform your vector and grow it on your plates. Does the EGFP source plasmid also have spec resistance? If not, you can eliminate gel purification entirely, which I would strongly recommend. Gel purification has low yield and can often damage DNA with UV exposure.

#4 och

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Posted 11 July 2012 - 12:53 PM

@ Nayeem991... Thanks for your reply. In fact I a new ligase and ligase buffer before doing the last ligation. But still the transformation still failed. Anyway, I will try your advise and check if the ligase and buffer are still in good condition.

#5 Nayeem991

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Posted 11 July 2012 - 08:04 PM

@ Och: You can also try to transform a small amount (may be 1 ul or even more less) of undigested binary vector. Then you would be able to understand whether there is any problem with the transformation step or the competent cell. In which bacterial strain you are trying to transform? If you could tell me the the transformation protocol you followed, i could suggest you any alternative... best of luck.

#6 och

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Posted 11 July 2012 - 08:45 PM

@ phage434... I appreciate your response. Can you please explain your second statement further. The EGFP source plasmid has an ampicillin resistance. i will like to know why you suggesting eliminating gel purification totally. Thanks a lot.

#7 och

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Posted 11 July 2012 - 08:54 PM

@ Nayeem991.. I am trying to transform into E.coli. below is the transformation protocol I have been using. I will also appreciate if you will explain the ligation ratio of vector to insert to me. As in, how do I calculate the different amounts of each to mix in a ligation mix. What I have been doing is to add more insert than vector. Thanks a lot.


1. Put 50ul aliquot of competent cell each into 3 tubes.
2. Put on ice immediately and thaw
3. Take 2ul ligation product and put into competent cell
4. Mix gently by pipetting
5. Let sit on ice for 45minutes
6. Heat shock at 42oC for 2 minutes
7. Put back on ice and incubate for 30minutes
8. Add 500ul SOC into tube
9. Shake at 37oC, 100rpm for 1 hour
10. Pour 100/200ul bacteria on LB + spc(50mg/l)
11. Incubate at 37oC overnight

#8 Nayeem991

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Posted 12 July 2012 - 02:46 AM

@ Nayeem991.. I am trying to transform into E.coli. below is the transformation protocol I have been using. I will also appreciate if you will explain the ligation ratio of vector to insert to me. As in, how do I calculate the different amounts of each to mix in a ligation mix. What I have been doing is to add more insert than vector. Thanks a lot.


1. Put 50ul aliquot of competent cell each into 3 tubes.
2. Put on ice immediately and thaw
3. Take 2ul ligation product and put into competent cell
4. Mix gently by pipetting
5. Let sit on ice for 45minutes
6. Heat shock at 42oC for 2 minutes
7. Put back on ice and incubate for 30minutes
8. Add 500ul SOC into tube
9. Shake at 37oC, 100rpm for 1 hour
10. Pour 100/200ul bacteria on LB + spc(50mg/l)
11. Incubate at 37oC overnight

Do you use whole 10 ul of your ligation mixture for transformation? You can use whole to increase the chance. you should check the efficiency of your competent cell by transforming undigested plasmid. Try again with controls (as i mentioned earlier), i hope you will be able to identify your problem. At the same time you can maintain following caution while preparing the ligation mixture.... add ligation buffer after vigorous vortexing (make sure your buffer is completely thawed and there is no undissolved residues after vortexing). Take the ligase from the surface (do not dip the tip into the enzyme). I don't know but i think these might help to overcome your problem. At least you would be able to identify the problematic step. Best of luck.
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#9 och

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Posted 12 July 2012 - 08:03 AM

Thanks Nayeem991. I will put all your suggestions into use and reply back to you.

#10 och

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Posted 12 July 2012 - 08:10 AM

@phage434. I now understand your point on the EGFP gene. Now, should I also gel purify the plasmid vector or not?

Thanks man.

#11 Nayeem991

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Posted 12 July 2012 - 08:17 AM

Another thing.... Take three plates and spread whole 500 ul bacteria (~150 ul in each plate) after growing in SOC... Please let me know after performing the experiment. I am eager to know the result.. I am giving you a final outline...
For ligation,
Tube 1: digested and dephosphorylated vector + insert (To get your desired clone)
Tube 2: digested and dephosphorylated vector and no insert (To make sure that your dephosphorylation is ok)
Tube 3: digested vector (not dephosphorylated) and no insert (To check your ligase or buffer is working well)
In addition transform undigested plasmid into the host to check the efficiency of your competent cell.

Regards
Nayeem

#12 och

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Posted 12 July 2012 - 03:01 PM

Thanks a lot Nayeem991. I will surely let you know when I perform it.
Do you think I can ligate gel purified binary vector DNA(spec resistance) with unpurified insert DNA (Amp resistance) and still get the gene to be cloned onto the binary vector?

#13 Nayeem991

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Posted 12 July 2012 - 10:11 PM

Thanks a lot Nayeem991. I will surely let you know when I perform it.
Do you think I can ligate gel purified binary vector DNA(spec resistance) with unpurified insert DNA (Amp resistance) and still get the gene to be cloned onto the binary vector?

I wont suggest you to use insert without purification. You can avoid gel run. You can purify the product after digestion. It will spare your DNA from UV exposure. Since the insert size is smaller than the original host vector (ampr), the chances of the insert is higher to be cloned and if there is any undigested vector remain in the digest you wont get it because of spec selection. Did you get my point? I think phage434wanted to mean it. if you want to use the digested product without purification, in that case you must inactivate the enzyme by heat treatment (650c for 10-15 mins) but don't use EDTA. usually 1:3 vector to insert ration is used in ligation. If you avoid gel run, you have to increase the amount of insert. It could be 1:10. In that case you may increase the final volume of your ligation mixture. You can make it 20 instead of 10.
Best of luck
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#14 och

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Posted 13 July 2012 - 05:50 AM

So how do I purify without gel run. do you a protocol for that?
Should I also do the same thing to the binary vector?

Thanks a lot.

#15 Nayeem991

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Posted 13 July 2012 - 10:03 AM

So how do I purify without gel run. do you a protocol for that?
Should I also do the same thing to the binary vector?

Thanks a lot.

which kit do you use for purification? I use promega gel purification kit. The protocol is as same as gel purification. In gel purification... we cut the gel weigh it and add equal volume of membrane binding solution (like.. 100 ul for 100 mg gel) and heat at 65. isnt it?
In case of purification without gel run... add equal volume of membrane binding solution ( 50 ul for your 50 ul digestion mix). If the volume of your digestion mix is 50 ul, you can add 50 ul ddH20 to make it 100 and then add 100 ul membrane binding solution (i always do this thing). then take it to column. the remaining procedure is same as gel purification. If you need not to isolate a particular band this procedure works well. I have done it several times and got very good result.

If you have any query regarding this, please let me know.

And yes i don't suggest you to do the same for binary vector. Because the undigested (if there is any) plasmid will give rise to false result unless you have blue white selection.

Edited by Nayeem991, 13 July 2012 - 10:06 AM.





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