iComment on what you expect to see on the LB Ampicillin Xg

i)Comment on what you expect to see on the LB + Ampicillin + X-gal plates containing ligation mix A – E (Presence vs. Absence of colonies, Blue vs. White colonies). Justify your answer.

ii) What can you say about the transformation experiment if the plate corresponding to transformation with Ligation mix E shows no colonies but colonies were found on other plates in the experiment?

iii)Which part of the experiment did not work if plate D has a large number of blue colonies? Justify your answer.

1.5 mL microcentrifuge tubes pipettor p20 and tips centrifuge pUC19 digested, no CIP pUc19, dephosphorylated DNA, digested T4 DNA Ligase (1 U/ul) 5X Ligase Buffer sdH20 Procedures: 1. Label 5 microcentrifuge tubes A-E 2. To each tube, add the various ligation components (in ul) as follows: A B C D E pUC 19 digested (your pUC/HII) 2 2 0 0 0 pUC 19 digested and dephosphorylated 0 0 2 2 0 lambda digested 0 2 0 2 0 5x ligase buffer 2 2 2 2 2 5 3 5 3 5 dH20 T4 DNA ligase Undigested pUC 0 0 0 0 2 3. Flick the bottom of the tube to mix the components, then briefly spin the samples in the centrifuge 4. Make sure you understand the significance of the reaction mixes. Tube A contains undephosphorylated pUC and ligase, but no DNA. If you compare the results of tubes A and B what do you learn? What do you learn from comparing tubes A and C? Tube C is a negative control for what? What do you learn from comparing tubes B and D? Which tube is the \"test\" (the one that should result in the desired colonies)?

Solution

1. Plate A, C and D will have untransformed blue colonies; Plate B will have transformed white colonies; E will not have any colonies.

2. Plate E does not have any colonies because it does not have the vector for Ampicillin resistance like other mixture.

3. If plate D has large number of blue colonies then digestion of the vector did not work properly. If the vector is digested correctly then the gene for the enzyme beta lactosidase will be cleaved and it would not have produced blue colonies.