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Somebody explain

  1. Jan 26, 2005 #1
    Somebody plz explain ...

    I have an oligo (A) (non-phosphorylated in 5') of ~ 100 bases.
    I have its complement (B) with two over hangs (~4 bases each) on both he sides(5' and 3').

    the overhangs on the B are palindromic. as in the 5' overhang is palindromic to itself.

    I annealed both of them.
    phosphorylated the sample.
    added ligase


    after ligation, when i ran it on the gel, i saw only monomers and dimers.

    Why is this so ???
    y not other multimers ???

    did i go wrong sumwhere ???

    plz help
     
  2. jcsd
  3. Jan 26, 2005 #2

    iansmith

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    I think the problem is that your B oligo has both overhang. I would have an overhang on each oligo so they annealed as follow (the overhang are in bold)

    Code (Text):
    [B]GCTA[/B]GCTGCAAAGCTGCTGC
        CGACGTTTCGACGACG[B]CGAT[/B]
    Also what is the annealing condition? You temperature might the optimal for annealing your primer.
    What kind of ligation process are you using? 4 C overnight or 25C for 1 hour. The overnight incubation increase the yield and the number of different products.
    Did you heat your DNA at 65c prior to adding your ligase and ligase buffer? This increase the odds of DNA coming together properly.
     
  4. Jan 26, 2005 #3
    the sequences are like :

    Seq A XX---XX

    Seq B GATCXX---XXCGAT

    so theoritically,
    they sud form like ...
    ------XX---XXGTACXX---XXCGATXX---XX
    GATCXX---XXCATGXX---XXGATCXX---XXCATG

    and so on....

    But it didnt !
    ligation was carried out at 25 deg for over night.
    sample was kept at 65 deg before adding ligase!
     
    Last edited: Jan 27, 2005
  5. Jan 26, 2005 #4

    iansmith

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    Hopefully you made a mistake will typing the text because your overhangs are not complementary.

    In theory, it should work but it is not always the case and having both overhang on the same oligo might make the physical interaction more difficults. :wink:

    That may be a stupid question but did you cool the sample prior to adding the ligase. Ligase is inactivated at 65C.
    The rest of the ligation appears to be ok.

    What are you annealing temperature and condition?
     
  6. Jan 26, 2005 #5

    Monique

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    first of all, the ends are not complimentary.

    karthik, which ligase are you using and how fresh is it? especially when the enzyme is atp dependent, you should make sure it's fresh since atp is prone to degradation.

    why do you use the phosphorylation step, can't you omit it?
     
  7. Jan 26, 2005 #6

    Monique

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    I also see a problem in the annealing step of the different oligos. It might be time consuming, but won't it be more efficient to clone the fragment of interest into a vector, grow it in e.coli and cut it out with restriction enzymes?

    That way you are absolutely sure that you have a double stranded piece of DNA with sticky ends.
     
  8. Jan 27, 2005 #7
    Sorry, I corrected it :biggrin:

    ------XX---XXGTACXX---XXCGATXX---XX
    GATCXX---XXCATGXX---XXGATCXX---XXCATG

    Annealed it at 37 deg for 3 hrs.

    I can clone it, but what i need is a tetramer...
    But i get only monomer and dimer :(
     
  9. Jan 27, 2005 #8

    Monique

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    so how sure are you that your enzyme is fresh and why are you doing the phosphorylation step
     
  10. Jan 27, 2005 #9

    iansmith

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    That might be the problem. Your annealing temperature is too low.

    I usully mix equimolar ratio of the primer in TEN Buffer (10 mM Tris, 1 mM EDTA, 0.1 mM NaCl, pH 8.0 with HCl). I then incubate the primer from 95C to 4C over 1 hour. I usually do this in a thermocycler.

    This might also be a suitable protocol

    http://micro.nwfsc.noaa.gov/forums/viewtopic.php?t=10003&highlight=oligo+annealing
     
  11. Jan 27, 2005 #10
    the dna that i use is of pnly ~ 100 bases. n its melting temp is 71 deg.

    Yes.
    I used fresh enzymes (NEB). i phosphorylated, since ligation needs phosphorylated ends ...
    The oligos provided do not have phosphate grp at 5'

    I first annealed the oligos at 37 deg for 3hrs.
    added T4 ligase buffer (since it has ATP).
    added PNKinase. left it for 1-3 hrs.
    deactivated at 65 deg for 10 min.
    then added T4 ligase and left it overnight at room temperature....
    the reaction volume is 15-40 uL.

    sud i increase teh annealing temp ?
    sud i decrease ligation temp ??
    is the reaction volume very low ?

    watever the mistake may be...
    y cudnt i see even a faint band of multimers ???
     
  12. Jan 27, 2005 #11

    iansmith

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    You have to increase your annealing temperature. I should be around 70C. 37C might created unspecific binding and it is suboptimal for binding. I suggest that you follow either protocol I gave you above.

    The rest is ok but you should not heat you ligase buffer to 65C. You should add the buffer after inactivation of the enzyme.
     
  13. Jan 27, 2005 #12

    Monique

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    karthik, did you try and increase the concentration of your oligos that you use during ligation? what concentration are you currently using?
     
  14. Jan 27, 2005 #13

    Monique

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  15. Jan 27, 2005 #14
    i have varied concentrations of the DNA. but of no use ...

    Can u temme wat happens to the buffer when it is heated to 65 deg ??
    will the ATP degrade ??/

    and i think i cant use 70 deg for annealing temp, coz the melting temp is 71 deg.
     
  16. Jan 27, 2005 #15

    Monique

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    between which concentrations did you vary it? you really need to know with what concentrations you are working.
     
  17. Jan 27, 2005 #16

    Monique

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    Ok, I looked up my experiment. This is what worked for me:

    9.0 ul DNA 210 bp [184 ng/ul]
    1.05 ul 10x ligase buffer
    0.5 ul ligase 3 U/ul

    I did a number of experiments to try and optimize the longer concatamer formation. What I found is that at timepoint 15 minutes, the reaction has already come to an end. Not sure why, most likely circular product formation. On gel I typically get a ladder of 6 bands and a smear on top where the fragments are too large to be seperated.

    The reaction also worked for DNA of 100 bp. I prepared the DNA by cutting it with restriction enzymes out of a vector, after which it was agarose gel purified.
     
  18. Jan 27, 2005 #17
    500ng/uL - 1ul, 2uL ... of oligo
    1000ng/uL(1 ug/uL) - 1ul, 2uL ... of oligo

    BTW,
    u still didnt temme wat wud happen if one heats the buffer to 65 deg ...
     
    Last edited: Jan 27, 2005
  19. Jan 27, 2005 #18

    iansmith

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    I don't know what happen per se but from personal experience, putting the ligase buffer at 65C always decrease my efficiency.


    The optimal annealing temperature is probably around 68C. I have also used annealing temperature above the melting temperate and it gave good results.

    The idea would be heat you oligos to 95C so they denature then cool let it cool down to room temperature so the the oligo anneal at the optimal temperature without you pick a temperature. That the principle in the two protocol i gave you.
     
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