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No Colonies on subcloning - (Apr/13/2006 )

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Folks,

I am trying to clone something since past 4 months...

After fruitless try of PCR segment cloning, and 2 RE digestion
and all possible ligation times n ratios.

I did TA cloning, Insert into TA vector, And Digested from one vector and
ligated to the other. with just one enzyme digestion.

Insert (0.9KB) Vector (5KB)
ligation 3hr n overnight
ratio 1:1, 3:1, 10:1

All kinda heat shock variations,

Ligase is fine n tested
Cells are brand new (One shot TOP10- Invitrogen) n give apt positive controls...

sad.gif(
Iam loosing confidence...

can anyone suggest anything?

-avt_09-

Can you digest with 2 enzymes (i.e directional cloning)?

-yata-

no colonies at all, or no positive colonies?

are you sure your media/antibiotics and stuff are OK?

no colonies at all is a bad thing sad.gif

are you running gels to check that you have good amounts of DNA at all the steps up to ligation?

have you sequenced the TA product to be sure it is what you think it is? (if I am understanding you correctly, you have made a PCR product, cloned it into TA, and are now popping out the insert and attempting to put it into another vector...is this right?)

good luck

-aimikins-

thanx sad.gif

I think I need to find some God/Godess of cloning and please them...

@Enthusiast

MAN!!
not even background!
whats the point using 2 enzymes

-avt_09-

i run gel after purification of digestion
one single clear band

i run gel after ligation
shows me decent bands!

I did not sequence the TA cloned insert
as I adopted this simple way 2 weeks before!
and digestion gel shows me fine results.

Cells are fine as they give positive control results

-avt_09-

Are u sure your vector has antibiotic-resistant genes? (for your +ve control are u using Kit's vector?)
Have u tried other vectors?

Are u using invitrogen or other kit? We had problems with invitrogen, and switched to Qiagen

-yata-

How did you prepare your vector to receive the insert? If you used CIP, you may have done so too aggresively and destroyed the ends.

How do you perform the ligation? What ligase, what buffer? How do you know your ligase is good (or the ATP in your buffer) is good?

-HomeBrew-

My +ve control was with puc19, the invitrogen given DNA.

My Ligase buffer is fine; however I add FRESHLY made 10mM ATP.

the other person in lab used these reagents and got the clone..

Well, vector has ampicilin resistance...

I donot treat with CIP
so damaging ends is out of question

-avt_09-

I think the key is that you're not even getting background, if you use only one enzyme and you don't CIP...if someone else uses all the same ligation reagents and gets it to work, then it must be after the ligation step that the problem occurs

are you SURE your media is OK? your antibiotic was added at the proper concentration? have you tried an alternate batch of plates/antibiotics?

and what about your cells? you got some colonies on the supercoiled DNA ligation control? have you done an efficiency check? you know, of course, that the products of a ligation reaction are going to transform at a much lower efficiency and probably at a lower concentration as well...if your already know all this, please don't get irritated with me I am trying to brainstorm tongue.gif

how many ul of your ligation mix are you adding to your cells? how many ul of cells?
how are you treating the cells post-transformation? how are you plating, and how much? please give as much detail as possible

-aimikins-

the ligation step that the problem occurs
but my gel shows me ligation bands of apt size sad.gif

are you SURE your media is OK? your antibiotic was added at the proper concentration? have you tried an alternate batch of plates/antibiotics?
yea its fine my TA cloning worked fine in this things and same cells


and what about your cells? you got some colonies on the supercoiled DNA ligation control?

yes


have you done an efficiency check? you know, of course, that the products of a ligation reaction are going to transform at a much lower efficiency and probably at a lower concentration as well..

ya i let the plates be in incubator for 18hrs

.if your already know all this, please don't get irritated with me I am trying to brainstorm tongue.gif

Any kind of storm is fine if it helps tongue.gif


how many ul of your ligation mix are you adding to your cells? how many ul of cells?
5ul i.e. 50 ng
ppl with 100ng get their work done
so i dont think its excess

how are you treating the cells post-transformation?

i add ligation let them be on ice for 30 min
heat shock for 90sec
tried 60 sec as well
how are you plating, and how much?
everything, with the help of beads that i spread them on plate


thankx for ur interest n concern smile.gif

-avt_09-

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