Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Did my EcoRI restriction digestion work? picture included! - (Jun/27/2012 )

Pages: Previous 1 2 3 4 5 Next

It should not matter if you put in the sequence or its reverse complement. Probably you are not doing the reverse complement correctly if you get a different number of cut sites when switching from one to the other.

-phage434-

phage434 on Mon Jul 2 13:26:35 2012 said:


It should not matter if you put in the sequence or its reverse complement. Probably you are not doing the reverse complement correctly if you get a different number of cut sites when switching from one to the other.


Hello Phage,
Here is my cDNA sequence
atggcgtcgaagaggatattgaaggagctcaaggatctgcagaaggatccccccacatcatgcagtgctggtccagtggcagaggatatgttccattggcaagcaacaatcatggggcctaccgatagcccttatgctggaggtgtatttttggtttcaattcatttccctccagattatccttttaagcctccaaaggttgccttcagaactaaggttttccatcccaacatcaacagcaatggaagtatttgtctggatattcttaaggagcagtggagtccagcattaaccatatccaaggtcctgctgtccatctgctctctgttgacagacccaaacccagatgatcctcttgtacctgaaattgctcacatgtacaagactgacagggccaaatacgaaaccactgctcgtagctggactcagaaatatgcaatgggatga
Here is its complement
TACCGCAGCTTCTCCTATAACTTCCTCGAGTTCCTAGACGTCTTCCTAGGGGGGTGTAGTACGTCACGACCAGGTCACCGTCTCCTATACAAGGTAACCGTTCGTTGTTAGTACCCCGGATGGCTATCGGGAATACGACCTCCACATAAAAACCAAAGTTAAGTAAAGGGAGGTCTAATAGGAAAATTCGGAGGTTTCCAACGGAAGTCTTGATTCCAAAAGGTAGGGTTGTAGTTGTCGTTACCTTCATAAACAGACCTATAAGAATTCCTCGTCACCTCAGGTCGTAATTGGTATAGGTTCCAGGACGACAGGTAGACGAGAGACAACTGTCTGGGTTTGGGTCTACTAGGAGAACATGGACTTTAACGAGTGTACATGTTCTGACTGTCCCGGTTTATGCTTTGGTGACGAGCATCGACCTGAGTCTTTATACGTTACCCTACT

If you enter each one in the following site (http://tools.neb.com/NEBcutter2/) you will realize that the complement has an EcoRI site while the cDNA sequence doesn't.

But I guess to know if my insert has an EcoRI cutting site, I would have to look at the cDNA sequence.
Thanks,
Yasamin

-Fluffy-

Fluffy on Mon Jul 2 17:25:15 2012 said:


But I guess to know if my insert has an EcoRI cutting site, I would have to look at the cDNA sequence.


Yes! Or, you look at the reverse complement...your complement sequence is from 3' to 5' and the EcoRI site is there if you read from 3' to 5' which is not correct and leads to misunderstandings

Altogether...your insert has no EcoRI site...that's why your fragment in the digestion is ~ 500 kb and not smaller...it would be if there was a EcoRI site.

Good luck for your further experiments.

-Papaver-

Just to be certain you've understood: You have taken the complement, not the reverse complement of the sequence. The complement will have different sites (and is unrelated to the original sequence). Only the reverse complement makes sense from a biological perspective, and it will have the identical restriction sites as the original sequence. You should review the anti-parallel structure of double stranded DNA to fully internalize this.

-phage434-

I am sorry I did not think you meant reverse complement. Thanks to you Papaver and Phage for the information. Phage, your last comment makes total sense and helps me understand things better. Papaver, since that is the case, I will try your protocol for digestion mentioned in earlier posts and let you know what happens.
Thanks again

-Fluffy-

Hello Papaver,
I did a digestion using the protocol you suggested and incubated for 1 hour at 37C. My picture is attached.
Attached Image
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!)

It still looks abit weird, what do you think?

Thanks again

-Fluffy-

Hey Fluffy,

I don't think it's weird..
Your EcoRI hasn't digested the Plasmids completely...except No. 9 of the digested sample. There you can see you fragment very nice and the plasmid part show only one rich band. The other digestions also show your insert although the bands are more or less weak. Furthermore you can see that all undigested plasmids move to the same size which implies that all contain the insert...

So everything is ok. I would take the plasmid/clone of the digestion No 9 and go ahead with the work. If possible sequence your insert to make sure that there are no mutations in.

-Papaver-

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

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

And the last image is same as the above after gel extraction and clean-up
Attached Image
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 for anyone who can help, I know this is extremely long but wanted to make things clear as to what I did.

-Fluffy-

Fluffy on Thu Jul 12 16:50:33 2012 said:


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


Hey,
there is no need to worry. The concentrations after gel extractions are often that low but it's absolutely enough for ligation.
Use around 40 ng of your double digested vector, that's a good concentration to work with...less would be also ok. The molar ratio of vector vs. insert should be 1:3 to 1:5. pET14b is ~ 4.6 kb, your fragment ~0.5 kb. So I would use 2.5 µl of your vector, 5.5 µl of your insert, 1.5 µl of ligase buffer (10x) and 0.5 µl ligase (I usually set up this reaction without using additional water and fill up final concentration with vector and/or insert). For transformation I would use 5 µl.
I'm only wondering why you digested your vector sequentially. If both enzymes are compatible with the buffer you can do this in one reaction as you have done with the Topo vector. I have also never dephosphorylated my vector before...even not when I digested with only one enzyme (but that's my way ;)
But anyways...just keep doing you stuff....that's fine :)
Cheers

-Papaver-

Im back Papaver for more not so well though..
As you know I continued with my ligation rxns.
I did four reactions for this matter. 3 are considered control reactions.
My protocols for the reactions are as follows:
Rxn1: Undigested Vector
Undigested pET 4b vector 2.5uL
5X ligase buffer 1.5uL
H2O 6uL
Rxn2: Double Digested Vector
DD pET 14b vector 2.5uL
5X ligase buffer 1.5uL
T4 DNA ligase 0.5uL
H2O 5.5uL
Rxn3: Double Digested Insert
DD insert 5.5uL
5X ligase buffer 1.5uL
T4 DNA ligase 0.5uL
H2O 2.5uL
Rxn4: 1:3 to 1:5 ligation rxn
DD pET14b vector 2.5uL
DD insert 5.5uL
5X ligase buffer 1.5uL
T4 DNA ligase 0.5uL

All reactions top up to a final volume of 10uL

I transformed my reactions into standard competent Bl21 cells (1uL of each ligation into 20uL Bl21 cells), put in water bath (42C) for 30 sec, then for one hour at 37C at a speed of 250rpm in a shaking incubator. I plated into ampicillin plates (25uL for plating).
I left overnight at 37C incubator.

Today I found not such a great results.
Rxn 1 plate: many colonies (makes sense!)
Rxn 2 plate: 5 colonies only (I believe I should not get any colonies on this plate)
Rxn 3 plate: No colonies (makes sense!)
Rxn 4 plate: No colonies (it is suppose to be my ligation reaction, but failed I guess)


I thought I should mention that my ligase is expired (expired July 2011), also the ligase buffer is 5X not 10X.

What do you think?!
Thanks Dear!

-Fluffy-
Pages: Previous 1 2 3 4 5 Next