2. Look at the attached vector map. Notice that there are two different cloning
ID: 256989 • 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
Answer 2. a] Restriction enzyme for MCS 1 is KpnI, EcorI and for MCS 2 BamBI, HindIII
b] It is not necessary that we have to be picked two restriction site per MCS, its only because picking of two restriction site save the tiem for restriction digestion.
c] it is often necessary to make sure cloning gene will go into the vector in frame because we are only interested in the desired gene if it does not go into vector then there is no use of using vector, as vector carry our gene of interest into the host. So there are so many maker for assuring that our gene of interest is inserted.
D] Steps involve in cloning gene of interest are
1. Tke out the genome of organism, who gene of interest you want to clone,
2 Then diigest the genome with any restriction enzyme [ it should be kept in mind that vector must have that resctriction site too, which you are digesting the genome]
3. Digest the vector also with the same restriction enzyme used in genome digestion. [ you can use either two restriction site for digestion or one].
4. After digesting both Vector and genome with same restriction enzyme, prepare the ligation enzyme [ enzyme used in ligation of gene of interest to vector]
5. after ligating the gene of interest into vector. this vector is inserted into the host [ organism in which you want to clone the gene, i preferably use E.coli DH5alpha for cloning]
6. after inserting vector into organism grow it for overnight or depend upon the cell division time of choosed bacteria, use plasmid isolation of colony to get the gene of interest.
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