49 replies to this topic

[Fluffy]

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

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

[Papaver]

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.

[Fluffy]

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
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

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

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.

[Papaver]

Fluffy, on 12 July 2012 - 08:50 AM, 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

[Fluffy]

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!

Edited by Fluffy, 19 July 2012 - 05:55 AM.

[Papaver]

Hi Fluffy,

so first...if you buffer is 5 x , you would use 2 µl ; final concentrations should always be 1 x. You should also check if the buffer contains ATP (ligation reaction depends on it), otherwise you have to add it to the reaction. If the buffer is as old (and often reused) as the enzyme it might be better to add ATP.
Do you incubate your transformation mix prior to heat shock? If not, do this for around 30 min. After heat shock, add ~ 1 ml LB medium and incubate for at least one hour. I often incubate two. Try different amounts for plating...say 50 µl, 100 µl, 200 µl. Then pellet the remaining cells, remove a bit of LB and resupend the pellet in the remaining ~ 150 µl of LB. So you would plate all your cells. But I would do these "plating series" only for your ligation reaction

Considering your colonies you got with the DD-plasmid...maybe some plasmids weren't double digested in your reaction and so they self ligated...if there are only 5 I would not worry...it can always happen that you get colonies with empty plasmid even if you do a double-digestion cloning.

Cheers

[Fluffy]

Thanks Papaver for your reply.

I believe the buffer has ATP as one of its ingredients and it is as old as the enzyme. I am thinking of getting a new one Friday ( an enzyme with a new buffer to be extra careful, I might get the 10X so that way I will stick to the 1.5uL)

Also for my transformation I do incubate it on ice for 5 min before heat shock. After heat shock I incubate again on ice for 2 min. Also according to my protocol after that I add 80uL SOC medium to ensure better spreading, and then I put on 37 at 250rpm speed for 1 hour. Thought I should be more clear

So if I add SOC medium do you think I should still add LB medium ( I am guessing it has to have ampicillin antibiotic)?

Thanks again dear!

[Fluffy]

Also I forgot to mention for my reactions I incubated in a thermocycler at 16C overnight

[Papaver]

Fluffy, on 19 July 2012 - 07:42 AM, said:

Thanks Papaver for your reply.

I believe the buffer has ATP as one of its ingredients and it is as old as the enzyme. I am thinking of getting a new one Friday ( an enzyme with a new buffer to be extra careful, I might get the 10X so that way I will stick to the 1.5uL)

Also for my transformation I do incubate it on ice for 5 min before heat shock. After heat shock I incubate again on ice for 2 min. Also according to my protocol after that I add 80uL SOC medium to ensure better spreading, and then I put on 37 at 250rpm speed for 1 hour. Thought I should be more clear

So if I add SOC medium do you think I should still add LB medium ( I am guessing it has to have ampicillin antibiotic)?

Thanks again dear!

SOC Medium is even better than LB, and for both: no antibiotics before plating the cells on LB/Amp-plates.
You can always play a bit around with your transformation mix. Incubation longer on ice before heat shock would give you cells more time to "get in contact" with the DNA. You can also add more of your ligation mix...1 µl is a small amount.
You can also plate more...

[Fluffy]

Hello Papaver

I proceeded with ligations and transformations. Your suggestions made things easier for me. Thanks . I did colony PCR for the chosen colonies from ligation plates to check for recombinant plasmids with my insert ligated. The result is the following attached picture: The PCR involved the use of primers specific to my insert and it seems to be my correct insert size: 447bp long.

The next step my professor asked me to do a PCR to determine the correct insert orientation. I am not sure how this works and how PCR can be used to determine insert orientation. I understand that I have to use one primer specific to my vector (pET14b) located near the multiple cloning site, and a second primer specific to my insert. But I do not understand how it works.

I already designed my primers that are specific to my vector, I designed more than one.
Examples:
5'T G G A G C C A C T A T C G A C T A C G3'

5'T G G A G C C A C T A T C G A C T A C G3'

Do you have an idea about this method?
Thanks again,
Fluffy:)

[phage434]

For PCR to work, the two primers need to be facing one another.  Depending on the orientation of your insert, a primer on the vector and another on the insert will either face each other or be oriented in the same direction.  You can determine the orientation of the insert by checking which of these two PCR reactions work.  You could also do a similar check by restriction digest, if you can find one (or possibly a collection) of RE sites on your vector and insert which give distinct patterns on digestion.

[Papaver]

Hi Fluffy

since you have cloned your fragment into the vector using two different enzymes, the direction of the insert is given by the restriction sites. I suppose you have added the Nde site to the 5 prime of your insert and the Xho I site at the 3 prime end. Anything else would not make sense to me and that's why I would check a few plasmids isolated from your clones by restriction digest phage 434 has already mentioned. Besides this I would also sequence your construct to make sure your insert (or protein) is in frame.

[Fluffy]

Hi,

Hope everyone is well.

I did a PCR to confirm the orientation of my insert in the pET14b vector. I ran two PCRs, first using a vector primer and a forward primer of my insert, then the same vector primer and the reverse primer of my insert.
The PCR using my vector primer and forward primer of my insert should give me the correct orientation of my insert only if I get a band of size 600-700bp. I attached a gel image showing bands of approximatly that size.


The PCR using vector primer and the reverse primer of my insert gave me a weird pattern


What do you think? I would assume my insert is in the correct orientation since the first picture gave clear bright bands.
Just a reminder the vector primer is a forward primer that was designed approx. 200bp-300bp of the multiple cloning site.

Should I move on?
Thanks again for all help!

[phage434]

I agree, your insert is in the direction amplified by your forward primer.  The weird bands are trash products resulting from long extention times and low annealing temperatures priming to who knows what.

[Fluffy]

Sounds good Phage434
I will proceed with my work then!
Thanks again