Question

8. You try to ligate a cDNA fragment with Bgl Il and Xho I sticky ends into the Bgl Il and Xho I sites in the multi- cloning site of a vector (vector plus insert: 7 kbp). The plasmid vector has an ampicillin resistance gene. What do you see on the plate next day if you spread the transformed bacteria on an LB agar plate with ampicillin- (1) bacteria with no vector, (2) bacteria with vector alone, (3) bacteria with vector plus insert, or (4) no bacteria? Put all numbers that apply. 9. What do you see on the plate next day if you spread the transformed bacteria on an LB agar plate with kanamycin-(1) bacteria with no vector, (2) bacteria with vector alone, (3) bacteria with vector plus insert or (4) no bacteria? Put all numbers that apply. 10. What do you see on the plate next day if you spread the transformed bacteria on an LB agar plate with no antibiotic-(1) bacteria with no vector, (2) bacteria with vector alone, (3) bacteria with vector plus insert or (4) no bacteria? Put all numbers that apply. 11. If the vector alone cut with Bgl Il and Xhol is 4 kbp, how big is the insert? 12. (1) Label the position of the positive and negative electrodes on the left of the box. Draw what the agarose gel would look like in the box below with the following lanes: 1 2 3 4 5 (1) 1 kb DNA ladder, from 1 kb to 12 kb, with 1 kb increments (2) vector alone, digested with Xhol and BamHI (3) vector plus insert, digested with Xhol and BamHI (4) vector alone, digested with BamHl only (5) vector plus insert, digested with Xhol only 13. Immunoglobulins consist of two heavy chains (50 kD each) and 2 light chains (25 KD each) joined by disulfide bonds. (1) After running an immunoglobulin sample on reducing SDS-PAGE there are _band(s), of the following size(s): (II) After running an immunoglobulin sample on non-reducing SDS-PAGE there are__band(s), of the following size(s): (iii) What is the molecular mass of immunoglobulin in: kD: daltons: What does one mole of immunoglobulin weigh in grams: (iv) How much immunoglobulin in grams would you need to make 1 mL of a 1 M solution? The correct answer is a number less than 1000, with no exponential notation and the correct prefix before g.

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Answer 1

8. Bgl II:
A^GATCT
TCTAG^A

Xho I:
C^TCGAG
GAGCT^C

Resistance marker on the vector = Ampicillin

1. No colonies on plate with bacteria with no vector

2. Since BglII and XhoI produce incompatible sticky ends, they cannot ligate together to produce self ligated vector.
So, the number of colonies on the plate will be almost zero (Assuming 100% restriction digestion efficiency)

3. Highest number of colonies can be observed

4. No colonies on plate with no bacteria