Recombinant DNA Bacterial Transformation

Student InstructionsLigation

Recombinant TransformationLigation

Obtain reagent tubes labeled: - pAmp (digested) - pKan (digested) - 10X LB/ATP (ligation buffer) - T4 Lig (ligase) - DW (distilled water)

Label a fresh sterile 1.5 ml tube “Lig” for the ligation reaction.

Recombinant TransformationLigation

Place the tubes marked “pAmp” and “pKan” in a 65oC water bath for 10 minutes.

Recombinant TransformationLigation

Use the matrix below as a checklist while adding reagents to “Lig” tube for the ligation reaction. Make sure to add the reactants in the order listed from left to right. Add all reagents directly into the solution. Use a fresh pipette tip

for each reagent.

Tube pAmp pKan DW 10X LB/ATP T4 Lig________________________________________________________ “Lig” 3 µl 3 µl 11 µl 4 µl 2 µl

Recombinant TransformationLigation

Incubate the reaction at room temperature for 24 hours.

IMPORTANT: Make sure you do not throw away the tube marked “Lig” when you dispose of your other

reagent tubes.

Recombinant TransformationLigation

If necessary, the ligation can be stored at -20oC after room temperature incubation.

Thaw the reaction before proceeding to transformation.

For the best results, the transformation should take place immediately after incubation (the next day).

Timing the events in this lab is very important.