3 replies to this topic

[jwkang]

My ligation conditions are as follows :
1. Vector preparation :
   A plasmid (5793 bp) digested by EcoRI & HindIII ----> 4512 bp  , 725 bp  , 509 bp , 47 bp.  The 4512 bp of DNA fragments are used as A vector.
   B plasmid (5871 bp) digested by KpnI & HindIII ----> 4606 bp , 1265 bp.  The 4606 bp of DNA fragments are used as B vector
   All of the vector DNA are purified from agarose gel.
2. Insert preparation
   PCR sequence (814 bp) with TA ligation ----> TA plasmid (4129 bp)
   TA plasmid digested by EcoRI & HindIII ----> 3248 bp , 793 bp.  The 793 bp of DNA fragments are used as A insert.
   TA plasmid digested by KpnI & HindIII ----> 3268 bp , 771 bp.  The 771 bp of DNA fragments are used as B insert.
   All of the insert DNA are purified from agarose gel.
3. Ligation reaction
   After confirming the vector and insert DNA by gel electrophoresis , I performed ligation reaction.
   Construct A : A vector + A insert
                 insert : vector = 1:1 , 3:1 , 5:1 , 10:1
                 DNA total amount = 40 ng ~ 200 ng
                 Total reaction volume = 20 uL
                 Reaction temp. = 4 degrees C overnight , 16 degrees C overnight , RT overnight
   Construct A : A vector + A insert
                 insert : vector = 1:1 , 3:1 , 5:1 , 10:1
                 DNA total amount = 40 ng ~ 200 ng
                 Total reaction volume = 20 uL
                 Reaction temp. = 4 degrees C overnight , 16 degrees C overnight , RT overnight
4. Transformation :
   ligation mixture : 10 uL
   Competent cells : 50 uL
   SOC medium : 250 uL
   Plating volume : 100 uL , 200 uL
   Plate : LB agar containing 100 ug/uL ampicillin
   There's no any colony growing on the plate after overnight incubation.
5. Gel electrophoresis of ligation mixture
   There are several signals of multimer form of vector which can be observed on the agarose gel. (>4.3 kbp)
   There is the signal of dimer form of B insert DNA but no multimer form of B insert observed on the agarose gel.(about 1.6 kbp)
   There is no the signal of dimer form and multimer form of A insert DNA observed on the agarose gel.
Could anyone answer my questions plz ?
I've done this experiment for 4 months but still not get the colonies.
Thanks very much !!!!
There is the attaching file to help you understand my experiments.

Attached Files

•  Construction of EGFP fusion DNA construct.pdf   819.79K   207 downloads

[phage434]

Almost always these problem revolve around transformation.  Tell us more about exactly how you are transforming and where the cells you are transforming come from, and how they are prepared.  10 ul of ligation is usually too much for chemical transformation, but I'd like to hear more about how you are going about it.

You can improve your post-ligation gel images by heat killing the ligase (assuming you are not using quick ligase buffers).
But most people don't bother with these gels.

[jwkang]

Thanks for your reply.
My competent cells are commercial high efficiency E. coli DH5alpha.
Here's my procedures.
->Thaw one tube(50 uL or 100 uL) of competent cells on ice.
->Pipet 10 uL ligation mixture into the cells. Mix them by gently tapping the tube.
->Incubate on ice for 30 min
->Heat-shock the tube at 42 degrees C for 40 sec
->Place on ice for 2 min
->Add 250 uL SOC medium and incubate at 37 degrees C with shaking 225 rpm for 1 min
->Spread 100 uL or 200 uL onto the LB agar plate containing the 100 ug/uL ampicillin
->Incubate overnight
Is there any problem in my protocols ?

[phage434]

It sounds pretty good, except I think you are using too much ligation product  I'd suggest 1-2 ul of ligation in a 50 ul transformation.  But I would think that with commercial cells, this is probably not the problem (assuming they have been stored correctly).

What volumes of DNA are you using in the ligation reaction?  High volumes of DNA can result in inhibitors of ligation coming from your gel purification.  I would try to make a ligation reaction with the majority of the volume being pure water.  This is much more important than high DNA concentrations.

You might want to revisit your gel purification reaction, and make sure that the column is spun dry to get rid of excess ethanol.

You might also be trashing your DNA with UV.  Try minimizing exposure, and using long wave (365 nm UV) or blue light for exposing the gels.

I'd recommend testing your competent cell efficiency.  Likely the cells came with dilute plasmid (10 pg/ul in TE).  Use 1 ul and expect 100-300 transformants.