Recombinant DNA Bacterial Transformation Student Instructions Ligation. Recombinant Transformation Ligation. Obtain reagent tubes labeled: - pAmp (digested) - pKan (digested) - 10X LB/ATP (ligation buffer) - T4 Lig (ligase)
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Obtain reagent tubes labeled:
- pAmp (digested)
- pKan (digested)
- 10X LB/ATP (ligation
- T4 Lig (ligase)
- DW (distilled water)
Label a fresh sterile 1.5 ml tube “Lig” for the ligation reaction.
Place the tubes marked “pAmp” and “pKan” in a 65oC water bath for 10 minutes.
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
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
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.