Long Adenine and Thymine stretches are also to be avoided as these will “breath” and open up stretches of the primer-template complex. Why would AT promote breathing?
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Long Adenine and Thymine stretches are also to be avoided as these will “breath” and open up stretches of the primer-template complex. Why would AT promote breathing?
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- What would be the consequence of inserting 10 random base pairs immediately downstream of the -10 box in a strong promoter? And What sort of lab techniques/experiments could be used to determine these consequences?The restriction enzymes KpnI and Acc65I recognize and cleave the same 6-bp sequence. However, the sticky end formed from KpnI cleavage cannot be ligated directly to the sticky end formed from Acc65I cleavage. Explain why.Many restriction endonuclease recognition sequences are palindromes. What are palindromes? Are the recognition sequences for AatII and DraI palindromes? Some restriction endonucleases produce blunt-ended pieces of DNA, while other produce DNA fragments with sticky ends. What is the difference? What type of ends do AatII and DraI produce?
- The restriction enzymes Kpn I and Acc 65I recognize and cleave the same 6-bp sequence. However, the sticky end formed from Kpn I cleavage cannot be ligated directly to the sticky end formed from Acc 65I cleavage. Explain why.Kpn I and Acc 65I are restriction enzymes that identify and cleave the same 6-bp sequence. The sticky end created by Kpn I cleavage, on the other hand, cannot be directly ligated to the sticky end formed by Acc 65I cleavage. Please explain why.The 3′-exonuclease activity of E. coli DNA polymerase I was found to show no discrimination between correctly and incorrectly base-paired nucleotides at the 3′-terminus; properly and improperly base-paired nucleotides are cleaved at equal rates there. How can this observation be reconciled with the fact that the 3′-exonuclease activity increases the accuracy with which template DNA is copied?
- Will restriction sites for an enzyme that has 4 bp in its recognition sequence be closer together, farther apart, or similarly spaced, on average, compared with those of an enzyme that has 6 bp in its recognition sequence? Expalin your reasoning?a.) wanting to clone gene Z into pVector, the gene is amplified by PCR and restriction sites are added to the flanking ends. without realizing that the antibotic resistant gen )tetR) had a Sal1 site, you decide to add EcoR1 and Sal1 recofnition sequencen into the 12-nucleotide primers. Write the sequence of the 2 primers, noting the 5' and 3' ends?Analyzing Cloned Sequences A base change (A to T) is the mutational event that created the mutant sickle cell anemia allele of beta globin. This mutation destroys an MstII restriction site normally present in the beta globin gene. This difference between the normal allele and the mutant allele can be detected with Southern blotting. Using a labeled beta globin gene as a probe, what differences would you expect to see for a Southern blot of the normal beta globin gene and the mutant sickle cell gene?
- What would three possible reasons be that a PCR wouldn't work after cloning a TAQ sequence into a His-tag vector making three different constructs and verifying via SDS-PAGE that the TAQ protein is there?Nonhomologous end-joining (NHEJ) of a doublestrand break almost always results in perfect resealingof the DNA lesion, without the loss or gain of nucleotide pairs. Yet CRISPR/Cas9, which produces doublestrand breaks, is a highly efficient method of makingsmall deletions or insertions at the targeted site. Howcan you resolve this apparent contradiction?Describe the process of cloning a DNA fragment into theBamHI and PstI sites of the vector pUC18. How would youscreen for clones that contain an insert? and explain the process(steps) by drawing
