3 replies to this topic

[plasmamembrane]

Hello,

 

    I tried to clone a 1.1Kb gene into 5.7Kb vector but failed. Below is the protocol:

 

    The insert was generated by PCR harboring BamH1 and HindIII restriction site. The primers were designed to have extra 4 bases for efficient RE cutting. I gel purified the DNA and set up restriction digestion with both enzymes ( BamH1-HF and HindIII-HF from NEB in Cutsmart buffer ). After digestion, I gel purified again.

    The vector was prepared by digesting with both RE and purified by gel extraction. I saw a clear and sharp band on gel after RE but I did not check two REs individually.

 

    Ligation setup:

 

    80ng vector , insert/vector ratio: 3:1 ( evaluated by spectrophotometer at A260 )

    ligase 1uL ( NEB, regular ligase )

    reaction volume: 20uL

    ligate at RT for 1hr, heat inactivate for 10min at 65 degC

 

    Transformation:

    DH5 alpha, competency around 10^7, prepared by Inonue method

    heat shock: 42degC for 45 sec

    recovery: SOC for 45min

    2uL of ligation solution was used to transform E. coli

 

    Culture:

    O/N at 37 degC on LB plate with selective antibiotics

 

No colony recovered!

 

Is it possible that UV exposure ruined the DNA?

 

 

Need your help. Thanks a lot.

[phage434]

Test your PCR product for correct digestion. I'd recommend this:

 

1. PCR as before

2. Column cleanup of the PCR product (no UV or gel). You can run a gel with a sample to test for the correct product and a single band.

3. Save most of your product, but aliquot 60 ng or so twice for digestions (sample 1 & sample 2)

4. Digest sample 1 with BamHI; digest sample 2 with HindIII

5. column purify each sample (no uv)

6. split each sample in half

7. ligate half of each sample as before (no vector, just one of the digested samples)

8. Run each ligated sample along with the half which was not ligated on a gel

9. If ligation and cutting worked, you should observe a twice-length product on the gel. If not, that enzyme is failing to produce a ligation ready product.

10. If both enzyme digestions show double length product, digest the remaining PCR product with both enzymes and column purify.

[plasmamembrane]

Thanks for your opinion. I will try it tomorrow.

On NEB website, it stated that HindIII-HF can't be heat inactivated; and it has high affinity with DNA.

I am planning to clean up the restriction digestion product with Qiaquick PCR clean up kit. But due to the high affinity of HindIII-HF with DNA,

I wonder whether or not I can get rid of the enzyme. Is there any other alternative choice for that? such as PEG or sodium acetate/ethanol precipitation.

 

Thanks again.

[phage434]

The column cleanup will be just as effective as gel purification. Both involve resuspension in high concentration Gu-HCl (buffer PB or equivalent). As I suggested earlier, a different choice of enzymes would make things easier. EcoRI, XbaI, SpeI, PstI all can be heat killed, for example.