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Did my EcoRI restriction digestion work? picture included.

Did my EcoRI restriction digestion work? picture included. - Molecular Cloning Forum

Did my EcoRI restriction digestion work? picture included. - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 06-29-2012, 11:47 PM
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Default Did my EcoRI restriction digestion work? picture included.



Hello Everyone,

I digested using EcoRI to analyze my transformants. The vector, pCR 4Blunt-TOPO, is 3956bp (an online image shows where EcoRI cuts). My insert is approx. 460-490bp.
I used 4uL plasmid DNA in a 10uL reaction volume (my plasmid concentration is approx. 100ng/uL). I used 2uL buffer, 1uL EcoR1 enzyme, and 3uL water. I am not sure why I have multiple bands at the top of the gel, and whether my digestion worked? The image of my gel is attached.


The organization of the gel is as follows,
The first lane from left is the 1kb ladder
The next 3 from the left are undigested plasmid (control)
The next following 10 are the digested ones.

Thanks!
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Old 07-03-2012, 08:54 AM
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Default Re: Did my EcoRI restriction digestion work? picture included.

Hey yasamino,
it looks pretty messy but you can see the insert, although you have lots of undigested plasmid.
There are many ways to improve your digestions.
1. Load a digested empty vector, you need that in order to be 100% sure of successful cloning
2. Increase the total volume of your digestion. It is known that none restriction enzyme works well in limited volume. 10μl even for Minipreps is considered a small volume. I would go with at least 30μl of total volume in each miniprep and I would add 1U of EcoRI/miniprep in my mastermix.
3. Prefer O/N digestions
4. Always verify your successful cloning of an insert with additional enzyme/s.
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  #3  
Old 07-06-2012, 01:13 AM
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Default Re: Did my EcoRI restriction digestion work? picture included.

Thanks Derek.
I did a new digestion but I still got similar results. I attached an image of my new digestion.
The image is organized as follows:
-the first 10 on the left after the ladder are the undigested plasmid
-the next 10 are the digested (the first 5 correspond to the second 5 of the undigested ones, and the second 5 correspond to the first 5 of the undigested ones, sorry for the confusion this may cause!)

My protocol was:
6uL DNA (concentration: lowest being 120ng/uL and highest is 163ng/uL)
2uL 10X buffer H
0.5uL EcoRI enzyme
11.5uL water
totaling up to 20uL reaction volume
incubated the reaction in a PCR at 37C for one hour
Attached Images
File Type: jpg 07.05.2012 Undigested TOPO EcoRI digestion.jpg (14.7 KB, 7 views)
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Old 07-11-2012, 10:57 AM
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Default Re: Did my EcoRI restriction digestion work? picture included.

Your EcoRI digestion worked, although there was not 100% digestion at all! Yet is till worked and the lower bands <500 bp reveal your successful cloning.
Explanation now of the bands.
In the undigested and digested ones you see a pattern of bands that is continuous.
The two faint higher bands are multimers of your plasmid.
The immediate lower your open circular plasmid.
Lower than the circular plasmid is the digested (linear) and under that runs the supercoiled plasmid.
In your digested minipreps you see that last band around 500bp, which is ofc the EcoRI - EcoRI insert.

More details can be found in the following link:
[Only registered users see links. ]

Savor it!
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  #5  
Old 07-12-2012, 05:02 PM
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Default Re: Did my EcoRI restriction digestion work? picture included.

Ok I proceeded with my cloning work.

After checking for my insert using EcoRI digestion, my next step to do was to digest my TOPO+insert recombinant with NdeI and XhoI enzymes to free my insert with 5' overhangs as result of the double digestion using the NdeI and XhoI enzymes.


My protocol for this part is as follows:

3ug of DNA (concentration being around 150-160 ng/uL)

3uL 10X buffer H (both enzymes compatible with this buffer)

1uL NdeI

1uL XhoI

XuL water

top up reaction volume to 30uL

Incubated digestion at 37 in a thermocycler for 4 hours.



My vector which I will subclone my insert into was also double digested with NdeI and XhoI.

I performed a sequential digestion where NdeI was added first, checked on gel after 1.5 hours, then XhoI was added.

I also single digested the same vector with NdeI only for later purposes to be used as a control in my ligation reactions.

Total incubation time was 4 hours as well.



The protocol for my sequential digestion of pET vector is as follows:

3ug of pET 14b vector (0.5ug/uL)

3uL 10X buffer H

1uL NdeI

1uL XhoI

XuL water

top up to 30uL



The single digestion is exact as above protocol, but no XhoI was added.



For the pET 14b vector digestion, a dephosphorylation reaction was also done. I used a 0.2U/uL calf intestial alkaline phosphatase, and I added 0.5-1uL of the enzyme to my digestions before running it on a gel. I incubated at 37C for 5 min. Then to inactivate the phosphatase I added EDTA to an equal final concentration of MgCl2 (found in buffer H) and incubated at 65C for 15 min.



For all the above work, after running on a gel I seem to get clear bands and seem to be at the right size. But after gel extraction and purification I obtained really low concentrations and very very faint bands (not seen on gel image especially for my insert). My concentrations for my insert were as low as 5.8 and 6.8ng/uL. My vector concentrations were 15.7ng/uL for the single enzyme digested one and 23.3ng/uL for the double digested one. I am worried to proceed to ligation with such low concentrations what should I do? By the way my gel extraction purification is a kit from Promega called Wizard SV Gel and PCR Clean-Up System



To refer to gel images
In order from first attached to last

#1
For the sequential and single enzyme digestion of pET 14b with NdeI
The gel is from left to right: 1kb ladder, NdeI single digested pET 14b, NdeI sequentially digested pET 14b, undigested pET 14b.


#2
For the TOPO+my insert NdeI and XhoI double digestion, pET 14b single digestion, and pET 14b double digestion NdeI and XhoI

The gel is from left to right: 1kb ladder, TOPO+my insert dble digestion, TOPO+my insert dble digestion, single digested pET 14b, dble digested pET 14b


#3
And the last image is same as the above after gel extraction and clean-up

The gel is from left to right: same as above. As you can see the first two bands are not visible (they were a bit visible as I played with contrast of image, but almost not to be seen)

Thanks Drekketh for your help again.
Much appreciated!
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