Homework Answers
Question 4.
1) EcoR1 and BamH1 will be used to insert our gene of interest into the MCS of pUC19. The correct orientation for cutting at beginning and end of insert is HindIII, SphI, PstI, SalI, XbaI, BamHI, SmaI, KpnI, SacI and EcoRI.
2) 3128 bp
3) Ampicillin resistance
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4. The vector below is called PUC19. It has a Polylinker site, also called a multiple...
3. Below is a diagram of the pUC19 cloning vector (plasmid): Sall EcoRI Hinc Il Kpnl...
3. Below is a diagram of the pUC19 cloning vector (plasmid): Sall EcoRI Hinc Il Kpnl BamHI Accii Pst! Hind III T1 Sacl Xmal xbal BSDMI Sphi Smal Polylinker lacz -ori PUC19 amp Fig_18-08 Genetics, Second Edition 2005 WH Freeman and Company a. Describe briefly the purpose of the: ori: ampR; lacZt: Palxlinker b. When bacteria containing this plasmid are grown on media containing x-gal and ampicillin, why do sometimes you see blue colonies and sometimes white colonies? What does...
The extracted plasmid DNA is pUC19. Look up the plasmid map and include in your pre-lab...
The extracted plasmid DNA is pUC19. Look up the plasmid map and
include in your pre-lab as well as your report.
a. What is the location of the BamH1 restriction site? Xmn1
restriction site? (sequence location by number). How many of these
sites exist each?
b. If single digestions were performed (one restriction enzyme)
with BamH1 and Xmn1, respectively, how many fragments would form,
and what would be the sizes of each of these fragments?
c. If a double digestion...
You are tasked to clone the human actin gene (blue) from a cDNA clone that you...
You are tasked to clone the human actin gene (blue) from a cDNA clone that you obtained from a collaborator into the cloning vector termed pUC119 using the HindIII restriction enzyme. The 4 blue arrows show the HindIII recognition sequence on the Actin cDNA clone. Plac MCS Hindill Sphl sbil Pst BspMI Accl Hincli Sall Xbal BamHI Smal Xmal Acc651 Kpnl Eco53ki Sacl EcoRI lacz pMB1 ori M13 IG PUC119 3162 bps CDNA Clone Amp a. b. Figure 2: a....
QUESTION 11 a) The diagram below shows a typical plasmid cloning vector. There are three components ORI, amp and restri...
QUESTION 11 a) The diagram below shows a typical plasmid cloning vector. There are three components ORI, amp and restriction enzyme recognition sizes. Explain the roles of each of these three components and explain why each is important for cloning genes. 6 marks] ORI Hindlll Sphl Pstl Sall Restriction Sites Xbal BamHI Smal Kpnl Sac EcoRI amp ORI role: importance: amp role: importance: Restriction sites role: importance: b) A technique called RT-PCR uses the enzyme reverse transcriptase (RT) in combination...
Early gene cloning experiments involved insertion at one restriction site in the vector; for example, the insert would h...
Early gene cloning experiments involved insertion at one restriction site in the vector; for example, the insert would have and EcoRI site at each end, and he vector would be opened at an RI site prior to litigation. Under what circumstances would asymmetric cloning be desirable, with the insert having a different site at each end? Please only typed replies and provide source where information was obtained. Thanks
This is what I was given to solve the question. Nothing else and I don't understand...
This is what I was given to solve the question. Nothing else and
I don't understand anything at all.
5. You are tasked to clone the human actin gene (blue) from a cDNA clone that you obtained from a collaborator into the cloning vector termed pUC119 using the HindIII restriction enzyme. The 4 blue arrows show the HindIII recognition sequence on the Actin cDNA clone. piac CS Hindi Sphi Sofi Psti BspMI Aocl Hincll Sall Xhal BamHI Smal Xmal Acc651...
If the target DNA (the 3.266 Kb E. coli genomic Bam HI fragment) has the same restriction sites on each end, there are t...
If the target DNA (the 3.266 Kb E. coli genomic Bam HI fragment)
has the same restriction sites on each end, there are two possible
orientations for the target DNA to insert into the plasmid. The
following restriction enzymes would cut the 3.266 kb Bam HI genomic
fragment containing the RecA gene once or twice or not at all. In
the tables below, list the expected DNA fragment sizes for the two
possible orientations. Round the DNA fragment sizes to...
Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids...
Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids (now called a vector because it will carry your gene of interest) it is important to considering existing genes / DNA elements. If a site is in the middle of a gene, you could lose or destroy that gene. If there are multiple sites for an enzyme, when you paste them together, multiple possible outcomes can arise. This is undesirable, because it confounds verification...
Chromosomal and plasmid DNA can be cut into manageable pieces by restriction enzymes. Using agarose gel...
Chromosomal and plasmid DNA can be cut into manageable pieces by
restriction enzymes. Using agarose gel electrophoresis, the DNA
fragments can be separated on a gel, based on their lengths. In
order to see the fragments, a stain is typically added to the gel.
The size of each fragment can be determined by comparing each one
to a DNA molecular weight marker of known size.
Below is a map of pBR22 plasmid. The position and base pair
number of the...
3. Below is a diagram of the pUC19 cloning vector (plasmid): Sall EcoRI Hinc Il Kpnl BamHI Accii Pst! Hind III T1 Sacl Xmal xbal BSDMI Sphi Smal Polylinker lacz -ori PUC19 amp Fig_18-08 Genetics, Second Edition 2005 WH Freeman and Company a. Describe briefly the purpose of the: ori: ampR; lacZt: Palxlinker b. When bacteria containing this plasmid are grown on media containing x-gal and ampicillin, why do sometimes you see blue colonies and sometimes white colonies? What does...
The extracted plasmid DNA is pUC19. Look up the plasmid map and
include in your pre-lab as well as your report.
a. What is the location of the BamH1 restriction site? Xmn1
restriction site? (sequence location by number). How many of these
sites exist each?
b. If single digestions were performed (one restriction enzyme)
with BamH1 and Xmn1, respectively, how many fragments would form,
and what would be the sizes of each of these fragments?
c. If a double digestion...
You are tasked to clone the human actin gene (blue) from a cDNA clone that you obtained from a collaborator into the cloning vector termed pUC119 using the HindIII restriction enzyme. The 4 blue arrows show the HindIII recognition sequence on the Actin cDNA clone. Plac MCS Hindill Sphl sbil Pst BspMI Accl Hincli Sall Xbal BamHI Smal Xmal Acc651 Kpnl Eco53ki Sacl EcoRI lacz pMB1 ori M13 IG PUC119 3162 bps CDNA Clone Amp a. b. Figure 2: a....
QUESTION 11 a) The diagram below shows a typical plasmid cloning vector. There are three components ORI, amp and restriction enzyme recognition sizes. Explain the roles of each of these three components and explain why each is important for cloning genes. 6 marks] ORI Hindlll Sphl Pstl Sall Restriction Sites Xbal BamHI Smal Kpnl Sac EcoRI amp ORI role: importance: amp role: importance: Restriction sites role: importance: b) A technique called RT-PCR uses the enzyme reverse transcriptase (RT) in combination...
This is what I was given to solve the question. Nothing else and
I don't understand anything at all.
5. You are tasked to clone the human actin gene (blue) from a cDNA clone that you obtained from a collaborator into the cloning vector termed pUC119 using the HindIII restriction enzyme. The 4 blue arrows show the HindIII recognition sequence on the Actin cDNA clone. piac CS Hindi Sphi Sofi Psti BspMI Aocl Hincll Sall Xhal BamHI Smal Xmal Acc651...
If the target DNA (the 3.266 Kb E. coli genomic Bam HI fragment)
has the same restriction sites on each end, there are two possible
orientations for the target DNA to insert into the plasmid. The
following restriction enzymes would cut the 3.266 kb Bam HI genomic
fragment containing the RecA gene once or twice or not at all. In
the tables below, list the expected DNA fragment sizes for the two
possible orientations. Round the DNA fragment sizes to...
Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids (now called a vector because it will carry your gene of interest) it is important to considering existing genes / DNA elements. If a site is in the middle of a gene, you could lose or destroy that gene. If there are multiple sites for an enzyme, when you paste them together, multiple possible outcomes can arise. This is undesirable, because it confounds verification...
Chromosomal and plasmid DNA can be cut into manageable pieces by
restriction enzymes. Using agarose gel electrophoresis, the DNA
fragments can be separated on a gel, based on their lengths. In
order to see the fragments, a stain is typically added to the gel.
The size of each fragment can be determined by comparing each one
to a DNA molecular weight marker of known size.
Below is a map of pBR22 plasmid. The position and base pair
number of the...
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