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Recombinant DNA Bacterial Transformation Student Instructions Ligation PowerPoint PPT Presentation


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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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Recombinant DNA Bacterial Transformation Student Instructions Ligation

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Recombinant dna bacterial transformation student instructions ligation

Recombinant DNA

Bacterial Transformation

Student Instructions

Ligation


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)

- DW (distilled water)

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


Recombinant dna bacterial transformation student instructions ligation

Recombinant Transformation

Ligation

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


Recombinant dna bacterial transformation student instructions ligation

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


Recombinant dna bacterial transformation student instructions ligation

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.


Recombinant dna bacterial transformation student instructions ligation

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.


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