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I would like to design primers to add a poly T slip site in front of the inserted cDNA genes in the directional human cDNA library so that all 3 reading frames of the insert are expressed. This is necessary since translation will begin from the prokaryote promoter rather than from any eukaryote promoter present. Designing these primers is not as straightforward as I thought initially because the 3' end of the inserted genes will have a poly A tail. Therefore, both the 3' end and the 5' end of the inserted gene will be "infused" to the plasmid using oligos that contain a poly T site. As I add the poly T site to the genes initially, I need to make sure that both poly T oligos are not in the mixture together at the beginning. Otherwise these oligos may "prime" in the incorrect location. Insertion Site of Plasmid 3' end plasmid sequence: CGGGGTACGATGAGA 5' end plasmid sequence: TTGATACCACTGCTT In the kit, the 2nd strand synthesis is the last PCR that occurs before the In-Fusion, and the In-Fusion reaction does not make use of any oligos. Therefore, the last oligos the kit uses are during this 2nd strand synthesis. The kit uses the 5' PCR Primer II A and the 3' In-Fusion SMARTer PCR Primer at this step. The sequence of the 5' PCR Primer IIA is not given. The sequence of the 3' In-Fusion SMARTer PCR Primer is given and is shown below. 3’ In-Fusion SMARTer PCR Primer (12 μM) 5’– CGGGGTACGATGAGACACCA-3’ This primer does not contain any poly Ts because these were already added during a previous step. I e-mailed Clontech to obtain the sequence of the 5' PCR Primer IIA, but they would not give this sequence to me. They said this was proprietary information that could not be disclosed. Therefore, I won't be able to modify the in-fusion primers to add the poly T when the cDNA genes are included into the plasmid at first. I will have to do the in-fusion exactly the way the company intended. Then once my genes are already inserted into the plasmid, I can insert the poly T site just before the 15 bp of homology on the plasmid on the 5' end side of the genes as well as mutate the 15 bp so that they no longer contain a stop codon in any of the 3 reading frames. This can be accomplished by linearizing the plasmid with the 15 bp after and the 15 bp preceding it. Then I can do an infusion using primers that contain the 13 Ts along with the appropriate 15 bp of homology. In the case of the 15 bp downstream of the 13 Ts I will introduce a point mutation to remove any stop codons. Linearizing the plasmid At this stage, the genes will already have been inserted, and the plasmid just needs to be linearized. Insertion Site Lin2 GGTGACGAACCCTAGGAGATCTC 5' 3' Lin1 Order Sequence: TCGACCTGCAGGCATGCAAGC Lin2 Order Sequence: CTCTAGAGGATCCCAAGCAGTGG 58.7 C OM Tm TCGACCTGCAGGCATGCAAGC 58.1 C OM Tm 5' 3' The plasmid will be linearized with these two Lin primers. An In-Fusion reaction will then be performed using primers with 13 Ts of overlapping sequence. TCGACCTGCAGGCATGCAAGCTGCTTGGGATCCTCTAGAG TCGACCTGCAGGCATGCAAGC Need to find stop codons in this sequence in the direction that the Lac gene is transcribed. GAGATCTCCTAGGGTTCGTCGAACGTACGGACGTCCAGCT reverse CTCTAGAGGATCCCAAGCAGCTTGCATGCCTGCAGGTCGA complement Stop Codons TAG TAA TGA The bottom strand specifies the codons and contains one stop codon. What are all of the possible one base pair mutations I could make from the codon TAG? This strand specifies the codons TAA TAC TAT TCG TTG TGG AAG CAG GAG Stop Tyrosine Tyrosine Serine Leucine Tryptophan Lysine Glutamine Glutamic Acid I think Leucine codon would be the most innocuous codon to mutate to. Lin1 Performing In-Fusion Insertion Site TCGACCTGCAGGCATGCAAGCTGCTTGGGATCCTCTAGAG TCGACCTGCAGGCATGCAAGC = codon to be mutated Left Plasmid In-Fusion Site Right Plasmid In-Fusion Site CTGCTTGGGATCCTCAAGAGAAAAAAAAAAAAA 58.1 OM Tm GACGAACCCTAGGAGTTCTC 5' 3' 58.1 OM Tm TTTTTTTTTTTTTAGCTGGACGTCCGTACGTTCG 5' 3' ATNL1 primer ATNL2 primer ATNL1 primer order sequence: CTGCTTGGGATCCTCAAGAGAAAAAAAAAAAAA ATNL2 primer order sequence: GCTTGCATGCCTGCAGGTCGATTTTTTTTTTTTT TCGACCTGCAGGCATGCAAGC