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Problem with restriction digestion - (Sep/29/2005 )

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I have to work on overexpression of a kinase. I am doing cloning using PCR. The primers are from start(Forward) and end(Reverse) with adapters of NdeI (start) and XhoI (end). The vector is pTYB2 from NEB.
I was having problem cutting the ends of my PCR product (1.9 kb). Later I realized that it was due to less number of bases preceding the recognition sequence. So I cloned it in pGEMT-vector (TA cloning). I have three positive clones (confirmed by colony PCR) with the insert but none of them (Miniprep DNA) is getting digested with the enzymes NdeI and XhoI (the adapter sequences in the primers). Though the same is being cut by BamHI and giving the desired fragment (that lies within the insert DNA). I thought it might be due to some contamination in the Miniprep so I purified the plasmid preparation using Qiagen columns and set up another restriction with NdeI/XhoI but still there is no result. I don't know what is happening. Can anyone help in any way?

-berwal-

Possible problems:
Wrong buffer.
Your enzymes are dead (NdeI should cut in pGEM T as well).
Your primers don't actually contain those sites.
You used an elution buffer instead of water which messes up your digest.

-randy-

QUOTE (berwal @ Sep 29 2005, 04:04 AM)
I have to work on overexpression of a kinase. I am doing cloning using PCR. The primers are from start(Forward) and end(Reverse) with adapters of NdeI (start) and XhoI (end). The vector is pTYB2 from NEB.
I was having problem cutting the ends of my PCR product (1.9 kb). Later I realized that it was due to less number of bases preceding the recognition sequence. So I cloned it in pGEMT-vector (TA cloning). I have three positive clones (confirmed by colony PCR) with the insert but none of them (Miniprep DNA) is getting digested with the enzymes NdeI and XhoI (the adapter sequences in the primers). Though the same is being cut by BamHI and giving the desired fragment (that lies within the insert DNA). I thought it might be due to some contamination in the Miniprep so I purified the plasmid preparation using Qiagen columns and set up another restriction with NdeI/XhoI but still there is no result. I don't know what is happening. Can anyone help in any way?


Did you sequence thoses clones ? Sometimes during cloning some weird recombination (rare event) can occur and mess up the extrimities. You should perform a control digest with the same enzymes on a known DNA

Actually your pGEMT shoul at leat be linearized cause there'sa NdeI site in the polylinker do you see at leat linear plasmid in your digest ? If not I would think that the DNA is just not digestable

One explanation might be endonucleases produced by the recipient E.Coli host and not removed during the miniprep ! Did you use the Optional wash with the PB buffer ?

Pesji

-pesji-

You should also check to make sure BamHI is not in the vector and maybe fooling you into thinking you have the correct insert. When you do these digests, do you get back supercoiled DNA or do you see absolutely nothing?

-tap14-

Did you see linearization indicating that one enzyme may have worked in the double digest? If so it is likely that XhoI is cutting because the NEB catalog says that NdeI is sensitive to contamination from minipreps.

As posted above, you should make sure the enzymes work using some other plasmid to test

Don't use the column to re-purify, depending on protocol used the columns can leave alot of residual ethanol. Do a phenol chloroform extraction and reprecipitate then try again.

Use NEB4 for double digestion. If you used the pGEM T-easy there is no BamHI site in the vector, not sure for the pGEM-T vector...

hope this helps.... smile.gif

-beccaf22-

There are no BamH1 sites in the vector pGEMT. So I am sure that the BamH1 digestion is from insert only. What is surprising is that the Nco1 digestion is also only linearising the DNA and not releasing the insert which should have been the case.
I am also attaching the NEB cutter display of the two orientations of the insert in the T-vector. I'll be grateful if someone could help as I have run out of ideas on this one after putting in 3 weeks of work.

-berwal-

QUOTE (berwal @ Sep 29 2005, 09:46 PM)
There are no BamH1 sites in the vector pGEMT. So I am sure that the BamH1 digestion is from insert only. What is surprising is that the Nco1 digestion is also only linearising the DNA and not releasing the insert which should have been the case.

I am also attaching the NEB cutter display of the two orientations of the insert in the T-vector. I'll be grateful if someone could help as I have run out of ideas on this one after putting in 3 weeks of work.


rolleyes.gif
OK so let's resume

You have done miniprep and they can be cut with BamH1 who gaves the right insert you say
How did you do the BamHI digest did you made a double digest ? Or if it's a single digest then it doesn't mean that it's your right insert ! I don't see any BamHI on the Map you attached

I ask you whether you sequenced the plasmid with for example Sp6 and T7 primers !

Could you attached the sequence of your primers ?

Pesji

-pesji-

QUOTE (pesji @ Sep 30 2005, 01:53 PM)
QUOTE (berwal @ Sep 29 2005, 09:46 PM)

There are no BamH1 sites in the vector pGEMT. So I am sure that the BamH1 digestion is from insert only. What is surprising is that the Nco1 digestion is also only linearising the DNA and not releasing the insert which should have been the case.

I am also attaching the NEB cutter display of the two orientations of the insert in the T-vector. I'll be grateful if someone could help as I have run out of ideas on this one after putting in 3 weeks of work.


rolleyes.gif
OK so let's resume

You have done miniprep and they can be cut with BamH1 who gaves the right insert you say
How did you do the BamHI digest did you made a double digest ? Or if it's a single digest then it doesn't mean that it's your right insert ! I don't see any BamHI on the Map you attached

I ask you whether you sequenced the plasmid with for example Sp6 and T7 primers !

Could you attached the sequence of your primers ?

Pesji


OK I should have attached other picture.
BamHI was done as a single digest.
Also I must say that my plasmid preparation is showing presence of concatamers.

Forward Primer: 5'-GGACATATGCTGGGGGGCAGCGCCG-3'
Reverse Primer: 5'-GGTCTCGAGGGCAGGGAGCTCTCCG-3'

-berwal-

OK I should have attached other picture.
BamHI was done as a single digest.
Also I must say that my plasmid preparation is showing presence of concatamers.
[/quote]

OK that's make sense can you send the primers as a text file ?
You didn't answer the question Did you sequence the clones ?

You should always check your clones cause even if the Taq error rate is low there's still some chances to get some messed up clones believe me I'm doing this type of stuff for 20 years wink.gif

Let's start to look at the primers then will see the other hypothesis
pesji

-pesji-

[quote name='pesji' date='Sep 30 2005, 02:23 PM' post='26279']
OK I should have attached other picture.
BamHI was done as a single digest.
Also I must say that my plasmid preparation is showing presence of concatamers.
[/quote]

OK that's make sense can you send the primers as a text file ?
You didn't answer the question Did you sequence the clones ?

You should always check your clones cause even if the Taq error rate is low there's still some chances to get some messed up clones believe me I'm doing this type of stuff for 20 years wink.gif

Let's start to look at the primers then will see the other hypothesis
pesji
[/quote]

No Sir I have not yet sequenced the clone as it takes a lot of time(2-3 weeks) here at an institute closeby. We don't have a facility with us.

-berwal-

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