Restriction enzymes orientation

The plasmid DNA was then isolated and checked for the presence of the insert and for the correct orientation using restriction enzyme digestions and DNA sequencing. 

In this experiment, you will amplify a fragment of pBluescript II (a plasmid), which includes the multiple cloning site (MCS) of the vector (Fig. 24-2). The pBluescript II plasmid comes in the S/K form and the K/S form. These two plasmids are identical except for the orientation of the MCS (see Fig. 24-2). Using restriction enzymes and agarose-gel electrophoresis, you will determine which of these two plasmids was used as a template in the PCR reaction. The sequences of the two primers that will be used in the PCR reaction are shown under Supplies and Reagents. Primer 1 will anneal to positions 188 to 211 (5 to 3 ) on one strand of the plasmid, while Primer 2 will anneal to positions 1730 to 1707 (5 to 3 ) on the opposite sttand of the plasmid (Fig. 24-3). On amplification, a 1543-base-pair fragment of DNA will be produced that includes the multiple cloning site of the plasmid. fl (an isoschizomer of SacT) and Kpnl will then be used to determine whether the S/K or K/S form of the pBluescript II plasmid was used as a template in the amplification reaction. 

Use 10 pL portions of these DNAs for restriction analysis to determine the presence and orientation of inserts. The gene of interest must be inserted in the correct orientation for expression. By digesting with a restriction enzyme that cuts at one end of the inserted gene and a second enzyme that cuts in the vector (e.g., Bg/II), the orientation of the insert can be deduced. 

DNA from the resulting colonies is then screened for the correct cassette orientation (the ligation is bi-directional) by restriction enzymes or PCR. Once the properly oriented clone is isolated, a destination vector is born. 

Which of the following DNA sequences is likely to be cut by a restriction enzyme Only one strand, written in the 5 to 3 orientation is shown, but you should assume the opposite strand is present to form a duplex. 

Two clones, carrying a 5.2 kb insert in inverse orientations were isolated and mapped by restriction enzyme analysis. The two clones were used to construct deletion clones with the exonuclease Ill/mung bean nuclease system. Deletion clones containing the psbG homologons region were sequenced. 

Because the use of PCR very often results in the introduction of unwanted mutations, it was decided to construct die whole P putida pepK gene from these two clones without PCR. First, the insert fragments of both recombinant clones were isolated and fused in vector pGEM5Zf(+) (Promega). A plasmid with the two insert fragments in correct order and orientation was then identified by restriction enzyme analysis. This plasmid contains the complete P. putida pepA gene without any mutation. 

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