PowerPoint Presentation Recombinant DNA Bacterial TransformationStudent Instructions Ligation

1
Recombinant DNA Bacterial Transformation Student
Instructions Ligation
2
Recombinant Transformation Ligation

• 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.

3
Recombinant Transformation Ligation

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

4
Recombinant Transformation Ligation
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
5
Recombinant Transformation Ligation
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.
6
Recombinant Transformation Ligation
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.