代做Project Seven: Cloning, continued代做留学生SQL语言程序

Project Seven: Cloning, continued (20 points)

In this second part of a two-part project you will explore the basic principles of cloning including primer design and finding compatible restriction enzyme sites between a cloning vector and an insert. These are widely used techniques in molecular biology labs.

I. Review Project Six (Quiz, 5 pts) In particular, you'll continue working with the primer pair you designed in Project Six.

II. Primer Testing (8 pts)

Before we modify the primers (from Project Six) with the added cut sites, we'll test whether or not they work by doing an “in silico PCR” reaction using the UCSC Genome Browser.

Set "maximum product size" to 6000. Leave everything else as is.

· Set the “max product size” to 6000.

· For the “Genome” drop down menu, select “Human”.

· For the “Assembly” drop down menu, select “Dec 2013”.

· Set "Target:" as "GENCODE Genes".

· Click "Submit".

1.  Does the results page show the the gene you meant to amplify? If not, you need to figure out what went wrong and try again. (1 pt)

2.  How long is this PCR product? (1 pt)

3.  Click on the title of the first result. Provide the chromosome and nucleotide positions of this result: (2 pts) chr___; position____________________-_____________________

When answering the question below, remember that your forward primer base-pairs to the complementary sequence, so it will look just like your coding strand, but the reverse primer base-pairs to the coding strand. In other words, one of your bold regions will match the primer sequence, and the other bold region will match the complement of the other primer sequence. Look at the generic picture below for help in visualizing this.

4.  Copy and paste the CFTR mRNA from the NCBI GenBank record below. Highlight in bold the sequence corresponding to each primer (in one case the highlighted region will match the bases in the primer; in the other case, the highlighted region with correspond to the complement of the primer). (2 pts)

5.  On the same sequence, indicate (by highlighting or circling, for example) the start codon and the stop codon. Remove most of the lines in between to save space on the page.  (2 pts)

III. Designing restriction enzyme cut sites into your primers (7 pts)

Your primer pair will allow you to PCR amplify many copies of the sequence from a batch of cellular mRNA, but you also want to clone the sequence into your vector (plasmid) so that you can express the gene. Remember that none of your plasmid MCS sites matched the available sites in your sequence so you'll have to engineer cut sites into the 5' ends of your primers. You'll use KpnI at the 5' end of the sequence, and BamHI at the 3' end, which both leave "sticky ends".

6. For each restriction enzyme below, write the recognition site in its double-stranded form, placing a slash sign (/) at the cut site on each strand. Label the 5’ and 3’ ends. You can find this information by mousing over the enzyme name on the pFLAG-CMV-1 map. The first of four strands is completed for you. (3 pts, one for each of the other three strands-plus-cut-site, including complementary strands)

KpnI:  5’GGTAC/C 3’

BamHI:

Now you will add the RE sequence to the 5' ends of each of your primers from Project 6. However, for the enzymes to cut efficiently, you'll also need to add a couple more nucleotides to each end after that. Let's use "CC", so each pair of primers will begin with CC.

7.  Show the new primer sequences below, including the new cut sites. Be sure to include both the sense and anti-sense strand for each primer. Also, include the 5’ and 3’ labels to mark the orientation. (2 pts for each primer)

Forward primer:

Reverse primer:

A PCR reaction using these primers will result in millions of copies of the sequence, flanked by the enzyme sites. You can then cut the products with KpnI and BamHI, leaving sticky ends-- this will be your 'insert'. At the same time, but in another tube, you can cut the plasmid vector with the same two enzymes. Then you'll allow the insert to join up by its sticky ends to the matching sticky ends of the vector. Presto!