2. Look at the attached vector map. Notice that there are two different cloning

ID: 257286 • Letter: 2

Question

2. Look at the attached vector map. Notice that there are two different cloning sites

called MCS1 and MCS2 that flank an intron.

a. Pick four restriction enzymes, one pair from each MCS and list them

here(2pt)

b. Why is it necessary to pick two restriction enzymes per MCS? (1pt)

c. When cloning genes, it is often necessary to make sure it will go into the

vector in frame. Why do you NOT have to worry about the sequence being

in frame in this case (Think about what we are trying to accomplish. 1pt)?

d. List the steps to describe how you will finish cloning the gene of interest

(4pts).

Created with SnapGene® (6057) NotI (6020) Spel PfIFI Tth111I (5) (6016) Bmti PvuI (136) (6012) Nhel (6008) SphI Fsp (155) BsaAI - DraIII (412) (6002) PstI Sbfl (5977) Ahdl BtgZI (413) (5902) Apal (5898) PspoMI (5847) BstEII (5753) BssHII 6000 (5749) Mrel-SgrAI (5678) PasI (5623) Agel ocS terminator (5301) BsmBI (5279) Xbal (5273) BamHI (5267) HindIII (5262) BspDI Clal XcmI (1239) (5151) Pacl pKANNIBAL 6063 bp (4466) KpnI (4462) Acc65I (4456) EcORI 4450) PaeR7I- PspXI TIiI Xhol BtgI (1635) 4326) BmgBI EcoRV (1767) (4263) PshAI 4053) AccI (4027) HincII (3935) BsaBI (3856) StyI 3000 (3816) Scal (3682) Bsal (3664) Stul (3332) Mlul (3097) Not (3089) Sac (3087) Eco53kI BspQI SapI (2806) (3058) Sfil MscI (3052)

Explanation / Answer

A. MCS -1 (KpnI, XhoI).

MCS-2 (XbaI,ClaI)

B.To clone the gene of interest without any frameshift mutation we can take 2 restriction sites in each MCS where intron PDK shown in the vector will be taken out and our gene will be cloned in place.

C. The vector has its own promoter and terminator and if the sequence is cloned with in the given restriction site we don’t need to worry about it is in frame or not, it will be in frame only sureThe vector has its own promoter and terminator and if the sequence is cloned with in the given restriction site we don’t need to worry about it is in frame or not, it will be in frame only sure.

D. The cloning of gene of interest needs an

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2. Look at the attached vector map. Notice that there are two different cloning

2. Look at the periodic table and use the atomic number and mass number of carbo

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2. Look at the attached vector map. Notice that there are two different cloning

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