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Problem in PCR cloning


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#1 tanaka

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Posted 19 August 2004 - 06:57 PM

:o I am trying my experiment in cloning the IRES region of HCV (around 450bp). I amplify this fragment from patients' DNA with two primers containing the restriction site of SalI and BamHI. Then I try to clone them into the vector phRL-FL (Promega).
1. I use the qiagen extraction kit for the insert purification
2. The double RE reaction is done with BamHI Buffer as mentioned by NEB in 2 hours with 5units each ( I am also afraid of Star activity B) )
3. The quick ligation kit NEB, which I incubate at 22.5C in 5 minutes as recommended
4. I also dephosphorylate the vector after RE cutting by CIP.
5. For transformation, I use 100 mcl of DH5a E.coli competent cells

However, so far I coudnot get any colony from the plate !!! I am so upset. I am new in this field, so if you have any idea and experience, please help me

Thanks in advance

Tanaka :angry:

Edited by tanaka, 22 August 2004 - 07:04 PM.


#2 bitpas

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Posted 20 August 2004 - 05:46 AM

Hello!

I also had problems with a BamHI/SalI digestion once
SalI is quite a nasty enzyme, check its properties in the neb catalogue! (2002/03, page 242) It needs somehow quite a lot of additional bases if you cut near the End of your DNA Fragment (eg. primer), and it is not efficient at all when adding only the ususal 4-5bp
How do your primers look like?

I could suceed my cloning by doing an overnight digestion (double digest)
I would suggest you try this.

Otherwise if ligation is the problem, try to do overnight ligation at 16 or even, if it still doesnt work over weekend at 4 (sometimes that helps..) with a normal T4-Ligase and try to play around with Vektor-Insert ratio.

You dont need to dephosphorylate the vector since sal-bam is not compatible.


greetings

#3 tanaka

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Posted 22 August 2004 - 07:01 PM

Thank you very much for your help and your idea

Here are my primer sequences

sense: 5'-ACGCGTCGAC GGC GAC ACT CCA CCA T-3' (SalI site underlined)

antisense: 5'- GGATCC AAC TTR ACG TCY TGT GGG CG-3' (BamHI site underlined)

Now I reorder the primer without degenerated nucleotides and add 2 more nucleotide to the BamHI site. Will wait and see ;)

Concerning the ligation, I am using the Quick Ligation Kit from NEB.

Do you have any idea about this? Or I should extend the ligation time to overnight, 16C ?

Thank you in advance

#4 bitpas

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Posted 23 August 2004 - 02:21 AM

Most restriction enzymes dont cut if their sequence is located at the very end of your DNA-Fragment.
As a general rule always add between 4 to 6 bases at the end of your Primer after the Restriction site.
you dont need to have complementary bases, I normally just add "TTAA"

antisense: 5'- GGATCC AAC TTR ACG TCY TGT GGG CG-3' (BamHI site underlined)
This sequence won't be cut, i would suggest you try this:
5'- TTAA GGATCC AAC TTR ACG TCY TGT GGG CG-3'

In your case i dont think ligation is the problem. I dont know the Quick Ligation Kit from NEB. All i know is that "normal" T4-Ligase is most efficient at 16 for 12-15h.
I would try again with your Kit.

Good luck!

#5 InvisibleSurfer

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Posted 23 August 2004 - 06:02 AM

Hi there,

A few points:

1. Do always run RE products on a high percentage gel and cut out the fragment you are interested in. Do not directly clean up the RE reaction as there will definitely be in there a few uncut molecules that will give A LOT of colonies on your ligation plates.

2. Extend digestion time

3. Extend ligation time (at least 4h, 2h is marketing bs)

Good luck

#6 lyrezxl

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Posted 23 August 2004 - 05:34 PM

hi,
as bitpas mentioned ur antisense will not be digested by BamHI.
u can just simply do a TA clone then cut out the fragment with BamHI/SalI.
anyway, if u "coudnot get any colony from the plate", .... at least ur vector is very good, :rolleyes: u can use it again. only need to prepare the insert :lol:
good luck!!

lyre

#7 tanaka

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Posted 23 August 2004 - 11:24 PM

I was so confused, but your advices make sense! Now I do these things, and wait for the result.

1. Reorder the primer
2. Use high quality of agarose (which one is better, now I am going to use sigma), and change the loading buffer (afraid of nuclease contamination!), because some other fragments with good primer design were also not working well!.
3. Try to do TA cloning in some isolates to save the DNA

Anyway, I am still a newbie. :rolleyes:

Thankyou all

#8 tltan

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Posted 01 September 2004 - 08:09 AM

Hi,

One thing i note is that I think SalI uses its own supplied buffer from NEB and cuts at zero percent efficiency using buffer 1, 3 and 4 from NEB. It also requires BSA. Cutting at 50% using buffer 2.

Probably a TA clone will ensure that the insert is properly cut and i am pretty sure it will be easier to work with and amplify inserts.

Good luck!
:(




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