Reverse primers

A PCR mastermix contains water, buffer, MgCl2, dNTPs, forward and reverse primers... 

The template used for the PCR amplification is plasmid DNA obtained by a simple mini-prep (Sambrook and Russell, 2001). The amplification reaction mixture consists of 50 fil of PCR buffer (provided by USB with the FideliTaq DNA polymerase) containing 1.5 mM MgCl2, forward and reverse primers (0.5 [iMeach), the four dNTPs (0.2 mMeach), 0.025 U//(I of FideliTaq DNA polymerase (USB, United States Biochemical), and... 

AP = adaptor primer FP = forward primer RP = reverse primer... 

Always design the forward and reverse primers from a CG-free region to amplify the promoter region. In rare cases, one CG could be included in the primer sequence. However, that C could not be the last few bases of the primer and avoid it as the last base. 

In addition to these primers, a nested primer from a CG-free region should also be designed for the sequencing. However, the forward or reverse primer could also be used for the sequencing. 

PCR screen colonies or plasmid minipreps as usual with a vector-specific (e.g. T7) forward primer and your gene-specific reverse primer. 

Reverse primer for aptamer amplification in pGEM-3Z vector (P-22 pGEM, 22 bp)... 

The purified DNAs are used as templates for in vitro transcription reactions. Forward primer (P-40, same sequence as primer used during SELEX process) and reverse primer (p-22 pGEM). 

Transform the ligation product into E. coli cells and screen minipreps for inserts by digesting with EcoRI and SacII. Correctly ligated clones show an 850 bp band for the insert and a 3.4 kb vector band. Inversely oriented inserts produce 150 bp and 4 kb bands. Clones without insert show only the 3.4 kb vector band. Use M13 forward and M13 reverse primers for sequence confirmation. Prepare Maxipreps for one confirmed clone and test the functionality of the lox-STOP-lox cassette as described below. 

Transform the reaction into E. coli cells and confirm recombination by digesting miniprep DNA with Xbal. Cre-recombined plasmids that lost the stop cassette produce two bands of 460 bp and 2.9 kb. Nonrecombined plasmids produce bands of 1.3 and 2.9 kb. After sequence confirmation using the M13 forward and reverse primers, prepare a Maxiprep from one confirmed clone. 

Amplify an appropriate DNA fragment with an appropriate polymerase according to the manufacturer s instruction (see Note 1 and 6). Make sure to tag the RNA polymerase promoter site to the 5 end of the reverse primer see Fig. 1). 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

A mixture of more than one set of forward and reverse primers specific for the gene of interest will enhance the signal... 

Figure 3-1. Schematic representation of the polymerase chain reaction. The two strands of the template DNA are represented by the white and black bars. The upper (forward) and lower (reverse) primers are indicated by a hatched and solid white arrow, respectively. The 5 and 3 refer to the corresponding hydroxyl groups on the ribose residue in the DNA backbone, and indicate the directionality of the DNA. DNA polymerases synthesize DNA in the 5 to 3 direction.

Amplify genes of interest using a high-fidelity polymerase (such as Platinum Pfx polymerase) with primers that contain 5 extensions for LIC. For the pNHis vector, the forward primer uses the extension 5 GGTATTGAGGGTCGC, followed by the cDNA sequence starting with the second codon. The reverse primer uses the extension 5 AGAGGAGAGTTAGAGCCTTA. Note that a stop codon is added to this sequence so that the product does not contain additional amino acids encoded by the LIC sequence (see Note 4). 

Forty-eight variants of p53 were generated by inverse PCR using the wild-type p53 expression vector, pQE-80L H6-BCCP-p53, as template. Phosphory-lated forward primers bore the sequence variation at the 5 -terminus followed by 20-24 nucleotides ofp53 sequence. Unphosphorylated reverse primers were... 

Wild-type murine genotypic FcRn forward primer (FcRn-F) GGGATGCCACTGCCCTG reverse primer (FcRn-R) CGAGCCTGAGATTGTCAAGTGTATT... 

Human FCRN forward genotypic primer (hFcRn-F) AGCCAAGTCCTCCGTGCTC reverse primer (hFcRn-R) CT CAGAGAT GCCAGT GTT CC... 

---