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PCR and cloning


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#1 predoc

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Posted 18 February 2009 - 10:34 AM

I am cloning 2 sizes of my gene in two different plasmids.

Construct I: the vector has a XhoI and EcoRV site. I generated SalI and PvUII sites flanking my gene in oligos. PCRed, gel purified the fragment. I digested with SalI and PvuII and then ligated with the vector that I had digested with XhoI and EcoRV. I have checked over 20 clones, but don't see positives

Construct II: I generated a plasmid with my gene and a FLAG-HA tag on the N-term part of the gene. Now I want to use this vector, but genrate a fragment of my gene similar to the human gene(make my gene smaller) yet still have this N-FLAG-HA. So I designed a forward primer in the middle of the gene(the part that I want) and a reverse primer to the HA part. I PCRed using KOD, used a blunt ligation mixture from the Novagen blunt ligation kit. I have again checked many mnay clones, but I don't see positives.

Pleas ehelp!!! I have been doing this since New Years!! My boss will throw me out! :o

#2 perneseblue

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Posted 18 February 2009 - 03:01 PM

right there are many things that can go wrong. We need more information to diagnose the problem. Could you please tell us exactly what you did. Every detail.

1 - Sal has problems. See NEB for a list. If possible try not to use this enzyme in a ligation strategy.

2 - With that said, all restriction sites need to be skirted by a few bp before the site will be cut efficiently by the restriction enzyme. SalI site needs a minimum of 4bp flanking both ends of the restriction site.

3 - over exposure to UV can damage your DNA fragment. Exposure time should be measured in seconds not minutes

4 - gel purify your vector. This is very useful to remove denature vector DNA. Denature DNA will not digest but will transform. Bad DNA prep can have lots of denatured plasmid and will be a problem.

5 - distance between XhoI and EcoRV site in vector? How far apart are they? If they are too close the enzymes won't cut.

6 - How long did you digest the vector and the insert? Time? Could right down the formulation used.

7- how much insert DNA and vector DNA do you have (in ng/ul) and what kind of ligation ratios did you use. The vector and insert ratios are in terms of mol (number of molecules), not volume or mass. What ligation conditions did you use?

8 - T4 DNA ligase and ligase buffer goes off fairly easily, the enzyme in particular. Has anybody in the lab experienced ligation problems? I would recommend doing a test, run out some ligated vector + insert DNA onto a gel. If the ligation worked, you should see high molecular weight bands, in addition to your vector and insert bands

9 - personally I would check more than 20 colonies... 48. But given this is a two way, 20 is enough. Can you do colony PCR to check for insertion?

Construct II: umm... something doesn't feel right. Isn't N terminal of a gene the 5' end... thus forward primer. In any case... welcome to blunt end ligation x_x .... I have not been able to find the kit in question from your description. Could you write down its name?

Beware of UV if you gel purified... UV destroys DNA.

General blunt end ligation things to do... increase ligation time with a decrease in ligation temperature. If you are ligating at 16 C reduced the temperature to 4 C. Can you use PCR to screen your colonies? Since this is an insert into an empty vector, it should be possible to detect the difference.

How big is the insert? If it is sufficiently large, you can actually take the bacteria, mix it with some lysis solution and dye and run uncut plasmid DNA on a gel. with the empty uncut vector (from the no insert control) as a control.
May your PCR products be long, your protocols short and your boss on holiday

#3 94mkiv

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Posted 18 February 2009 - 06:02 PM

"Construct I: the vector has a XhoI and EcoRV site. I generated SalI and PvUII sites flanking my gene in oligos. PCRed, gel purified the fragment. I digested with SalI and PvuII and then ligated with the vector that I had digested with XhoI and EcoRV. I have checked over 20 clones, but don't see positives"

Is there a reason you couldn't digest your PCR product (add XhoI site into oligo) with the same enzymes you used for your vector?

XhoI ans Sal I have similar restriction site sequence but are not identical. It may help to use the same RE (XhoI or Sal I) for both the vector and PCR product.

Sal I is also not the most robust RE as mentioned. Did you check to make sure you used the right units and buffer?

Was the digested vector gel purified also?

Edited by 94mkiv, 18 February 2009 - 06:11 PM.


#4 predoc

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Posted 19 February 2009 - 06:50 AM

For Construct I: I need full length gene. I have a XhoI and EcoRV site in the middle of my gene. So I cannot use these sites. SalI needs NEB U and BSA. It has 50% activity in Buffer 3. PvuII works with all NEB buffers: 1,2,3, 4. So I used, Buffer 3 and BSA in the reaction mix. I digested for about 5 hours.

I did not gel purify the vector because the XhoI and EcoRV sites are back to back: CTCGAGGATATC. I did the ligation o/N at 4 C

This is a scond method I am trying for putting the construct in. What i want is to put my gene with a C-term FLAGHA in pUAST vector. I have the untagged version of my gene in this vector. I tried introducing the tag with PCR and it did not work. So I am now using another vector that has this tag. The cloning can only be done between the XhoI and EcoRV in this new vector. Once I have this going, I will recombine in my pUAST vector with my gene. So that's wat I need in the end.

Thanks

"Construct I: the vector has a XhoI and EcoRV site. I generated SalI and PvUII sites flanking my gene in oligos. PCRed, gel purified the fragment. I digested with SalI and PvuII and then ligated with the vector that I had digested with XhoI and EcoRV. I have checked over 20 clones, but don't see positives"

Is there a reason you couldn't digest your PCR product (add XhoI site into oligo) with the same enzymes you used for your vector?

XhoI ans Sal I have similar restriction site sequence but are not identical. It may help to use the same RE (XhoI or Sal I) for both the vector and PCR product.

Sal I is also not the most robust RE as mentioned. Did you check to make sure you used the right units and buffer?

Was the digested vector gel purified also?



#5 predoc

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Posted 19 February 2009 - 08:27 AM

Hi Pernese Blue: For Construct I:
1. I answer this in the next post.
2. I had to engineer a SalI site at the 5'end of my gene by oligos. i had atleast 10 bases on either side of SalI site.
3. exposure time to gel extraction was about 3-4 seconds
4. I did not gel purify the vector becaue XhoI and EcoRV are back to back. See in my response to the past after yours.
5. I digested both for 4-5 hours. Vector+EcoRV +XhoI +BSA in NEB buffer 2
Purified insert in NEB buffer3+SalI+PvuII
6. I approximately used the vector and insert ratio. I think I ended upwith 30ng of vector and 3 times of this of insert in the ligation mix. Ligation was at 4C o/n with T4 DNA ligase and buffer.
7. This is actually a second strategy I am using to put my construct in a vector. I need to put a C-term FLAG-HA in my gene. I have my untagged gene in a pUASt Drosophila based vector . I had successfully made an N-termFLAG-HA in this vector using long oligos. But i ahd trouble putting the tag C-terminally using my untaggedgene-pUAST vector. So I switched the strategy. I have a vector with a HA-FLAG tag. Using the XhoI-EcoRV sites, I can put my gene in this vector. Then I plan to recombine it into the untagged gene-pUAST vector I made a couple of months ago. But nothing seems to work.


For construct II, I want to make a truncated version of the gene. I need the second half of my gene. Since I have my gene with an N-term FLAG-HA, I was trying to put a forward oligo to the middle of the gene, the reverse oligo to the tag I made. I used 2 different PCR enzymes: KOD and Pfu Pol in two different reactions. I then gel purify the PCR product, blunt it with End conversion mix from the Novagen perfectly Blunt Cloning kit, ligate using T4DNA ligase, and transform in normal lab DH5alpha cells. But negative!! I have made 2 different forward oligos, but it doesn't work. The GC content is very repititious in the area I want to start the oligo.

Thanks a lot, pernese blue, for your time and patience. I appreciate this.

right there are many things that can go wrong. We need more information to diagnose the problem. Could you please tell us exactly what you did. Every detail.

1 - Sal has problems. See NEB for a list. If possible try not to use this enzyme in a ligation strategy.

2 - With that said, all restriction sites need to be skirted by a few bp before the site will be cut efficiently by the restriction enzyme. SalI site needs a minimum of 4bp flanking both ends of the restriction site.

3 - over exposure to UV can damage your DNA fragment. Exposure time should be measured in seconds not minutes

4 - gel purify your vector. This is very useful to remove denature vector DNA. Denature DNA will not digest but will transform. Bad DNA prep can have lots of denatured plasmid and will be a problem.

5 - distance between XhoI and EcoRV site in vector? How far apart are they? If they are too close the enzymes won't cut.

6 - How long did you digest the vector and the insert? Time? Could right down the formulation used.

7- how much insert DNA and vector DNA do you have (in ng/ul) and what kind of ligation ratios did you use. The vector and insert ratios are in terms of mol (number of molecules), not volume or mass. What ligation conditions did you use?

8 - T4 DNA ligase and ligase buffer goes off fairly easily, the enzyme in particular. Has anybody in the lab experienced ligation problems? I would recommend doing a test, run out some ligated vector + insert DNA onto a gel. If the ligation worked, you should see high molecular weight bands, in addition to your vector and insert bands

9 - personally I would check more than 20 colonies... 48. But given this is a two way, 20 is enough. Can you do colony PCR to check for insertion?

Construct II: umm... something doesn't feel right. Isn't N terminal of a gene the 5' end... thus forward primer. In any case... welcome to blunt end ligation x_x .... I have not been able to find the kit in question from your description. Could you write down its name?

Beware of UV if you gel purified... UV destroys DNA.

General blunt end ligation things to do... increase ligation time with a decrease in ligation temperature. If you are ligating at 16 C reduced the temperature to 4 C. Can you use PCR to screen your colonies? Since this is an insert into an empty vector, it should be possible to detect the difference.

How big is the insert? If it is sufficiently large, you can actually take the bacteria, mix it with some lysis solution and dye and run uncut plasmid DNA on a gel. with the empty uncut vector (from the no insert control) as a control.



#6 perneseblue

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Posted 19 February 2009 - 01:12 PM

I did not gel purify the vector because the XhoI and EcoRV sites are back to back: CTCGAGGATATC. I did the ligation o/N at 4 C


Given the closeness of the two sites, I would try recutting the vector, starting with XhoI followed by EcoRV. If XhoI were to cut first EcoRV would have only 1bp of skirting and would probably not be cut efficiently.

I would also gel purify the vector to remove any denature plasmid, gDNA and other contaminants.

Also give your ligase buffer a good sniff. It should smell strongly of DTT. If it does not, it is time to change the buffer.

Once the vector and insert have ligated, could you run out a sample of the ligation mix onto a gel (use a narrow well) to see if the DNA has been ligated. You should see high molecular weight bands.

And what is the volume of the ligation mix?

As for Construct II.... despite the template problems a PCR product is obtained, right? If that is so a the system should work. What kind of screening system are you using? When you don't obtain any positives does it mean you are detecting plasmids with contain an insert but not the right one. Or do you mean only empty vector, or no colonies obtained.
May your PCR products be long, your protocols short and your boss on holiday

#7 predoc

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Posted 20 February 2009 - 08:50 AM

Hi pernese blue,

I get smaller sized clones or empty vector fpr both constructs.

I am repeating the digestion right now for construct I. I am cutting them sequentially as u suggest. Vector with XhoI first and PCR product with SalI first. I am using 3 ul of vector at .125ug/ul conc and 8ul of PCR product of conc. 0.1ug/ul. I am first doing a 10ul reaction. When I add the second enzyme and buffer, I will scale upto 20ul. Then set up ligation. I will use 3ul(45ng) of ligation mix and 4ul(160ng) of PCR mix for a 20ul final ligation reaction. What do you think? Should i gel purify both first. Then I will have losses of DNA, but maybe I can set up bigger volumes for ligation????

#8 perneseblue

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Posted 20 February 2009 - 01:12 PM

I am a proponent for the philosophy of "work with enough DNA that waste is not a concern."

For Construct I, the sequential digest is okay. But I think you should cut more DNA, more vector and more insert. You should cut enough DNA that you will not be bothered by DNA loses.

The vector should be gel purified after the sequence digest but the PCR insert can be double digested with SalI and EcoRV. The PCR insert must be cleaned before digestion, firstly to remove the polymerase and secondly to remove free nucleotide which can inhibit SalI. Use PCR clean column clean up kit or use gel purification.

The ligation should be based on mol ratio of vector and insert. Not on mass.

As for Construct II,
- it is picking up truncated PCR insert?
If that is the case, the PCR may not be working. Have you visually confirm that the PCR product is being made? Is it the right size?
May your PCR products be long, your protocols short and your boss on holiday

#9 pcrboy

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Posted 20 February 2009 - 11:49 PM

I think the fact you have successfully cloned other vectors, suggests that your reagents are fine. I also doubt it is your ligation since its an overnight reaction. Also, I disagree with an earlier post that screening 20 clones is too small. How many clones are you getting from your ligation transformation? Do you have a transformation with vector-only for a negative control?

#10 predoc

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Posted 21 February 2009 - 05:45 AM

Hi pernese blue,

I ran the ligation mix this morning. I also ran uncut starting vector that has a size of 8.5 kb along with it. The new vector size should be 10.5 kb. I saw that the supercoiled starting vector is about the same size as the band I see for the ligated DNA. Do you think I have the insert in my vector?

Thanks

#11 scolix

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Posted 21 February 2009 - 07:06 AM

Hi pernese blue,

I ran the ligation mix this morning. I also ran uncut starting vector that has a size of 8.5 kb along with it. The new vector size should be 10.5 kb. I saw that the supercoiled starting vector is about the same size as the band I see for the ligated DNA. Do you think I have the insert in my vector?

Thanks



if both the uncut plasmid of both the ligated and starting vector run at the same level, then highly unlikely that the ligation worked.

I somehow have a feeling that the DNA is not getting digested properly. Is there possibility to use any other enzyme? Try to go over the sites.

What is your ligation recipe? How much DNA do you use? Also could you verify if the restriction enzymes are working optimally.

#12 predoc

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Posted 22 February 2009 - 06:13 AM

Hi Scolic,

My ligation recipe has about 120ng of digested vector and about 500 ng of digested insert(obtained from PCR). I ligate a total reaction of 20ul. I add 1 ul of T4 DNA ligase. The ligation is o/n at 4C.

Thanks a lot

Predoc....

Hi pernese blue,

I ran the ligation mix this morning. I also ran uncut starting vector that has a size of 8.5 kb along with it. The new vector size should be 10.5 kb. I saw that the supercoiled starting vector is about the same size as the band I see for the ligated DNA. Do you think I have the insert in my vector?

Thanks



if both the uncut plasmid of both the ligated and starting vector run at the same level, then highly unlikely that the ligation worked.

I somehow have a feeling that the DNA is not getting digested properly. Is there possibility to use any other enzyme? Try to go over the sites.

What is your ligation recipe? How much DNA do you use? Also could you verify if the restriction enzymes are working optimally.



#13 predoc

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Posted 22 February 2009 - 06:18 AM

Hi PCR boy, I spread the transformation reaction in 2 titers. The first one is 50ul of a 1050ul transformation reaction. On this plate I typically get 5-10 clones. In the second, I spin down all the remaining cells and plate all of them in 50ul. On this plate I get more than 20 clones. So I pick 10 at first. Check them and then pick 10 more from either plate. In all I have checked 20-25 each time I did this. I did the negative control only once when I started in Jan with the first technique of introducing the tag by PCR. I did it again yesterday. i will check my latest result and tell you in about an hour.

Thanks,
predoc


I think the fact you have successfully cloned other vectors, suggests that your reagents are fine. I also doubt it is your ligation since its an overnight reaction. Also, I disagree with an earlier post that screening 20 clones is too small. How many clones are you getting from your ligation transformation? Do you have a transformation with vector-only for a negative control?



#14 PMB_stu

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Posted 22 February 2009 - 09:55 AM

hi
i also faced the same problem while cloning PCR product directly.
Then I got an idea of first cloning that PCR product in any T/A cloning vector. Then from that you just go for digestion of insert.
I also suggest you to gel purify ur vector and insert.

Hope it'll work

#15 predoc

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Posted 23 February 2009 - 07:02 AM

I do have colonies on my negative control plate but when I plate the concentrated cell mix. Unfortunately, my ligation mix also gives me about the same number of colonies, more on the conc cells. Do you think EcoRV and XhoI need more digestion than 4-6 hours??

Thanks

Hi PCR boy, I spread the transformation reaction in 2 titers. The first one is 50ul of a 1050ul transformation reaction. On this plate I typically get 5-10 clones. In the second, I spin down all the remaining cells and plate all of them in 50ul. On this plate I get more than 20 clones. So I pick 10 at first. Check them and then pick 10 more from either plate. In all I have checked 20-25 each time I did this. I did the negative control only once when I started in Jan with the first technique of introducing the tag by PCR. I did it again yesterday. i will check my latest result and tell you in about an hour.

Thanks,
predoc


I think the fact you have successfully cloned other vectors, suggests that your reagents are fine. I also doubt it is your ligation since its an overnight reaction. Also, I disagree with an earlier post that screening 20 clones is too small. How many clones are you getting from your ligation transformation? Do you have a transformation with vector-only for a negative control?






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