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===polyA tailing (T7tspn-top3) Reference/Notes=== *going to try A tailing with PolyA polymerase first then RT with clontech primer. "3' SMART CDS Primer II A" from [http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/cDNA_Synthesis_Kits/Ultra_Low_Input_RNA_cDNA_Synthesis?sitex=10020:22372:US SMARTer Ultra Low Input RNA for Illumina Sequencing kit] *20 μl 200 μl 3’ SMART CDS Primer II A (12 μM) 5’–AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N–3’ (N = A, C, G, or T; N-1 = A, G, or C). Also synthesizing my own (rui has) since theirs is modified 25 μl 250 μl IS PCR Primer (12 μM)* 5’– AAGCAGTGGTATCAACGCAGAGT –3’ PolyA tail addition *using Enzymatics [http://enzymatics.com/product_p7460-l.htm polyA polymerase] 10X Poly(A) Polymerase Reaction Buffer (B7460): 500 mM Tris-HCl 2.5 M NaCl 100 mM MgCl2 pH 7.9 @ 25°C first and second strand synthesis first strand synthesis (MMLV RT) RNA 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNNNNNN CTGTCTCTTATACACATCTTAATGTCTCCCA(30) -3' 3'- N,N-1,T(30)CATGAGACGCAACTATGGTGACGAA 5' (clontech primer ) PCR/second strand synthesis *taq polymerase as 5' -> 3' exonuclease activity so will destroy primer in bottom strand synthesis *only issue is possible hairpin formation of ends since they are complementary, thus not allowing binding of T7-sssF2, but it will be seen. Tm of complemntary sequence. GGGAGACATTAAGATGTGTATAAGAGACAG Tm=60.71 Tm of primer is, AAGCAGTGGTATCAACGCAGAGT TM=61.02 *single insertions will not have a problem, this is only with adajacent insertions. (T7-top3-sss) 5'- GGGAGACATTAAGATGTGTATAAGAGACAG -3' 5’- GGGAGACATTAAGATGTGTATAAGAGACAG NNNNNN(NNNNNN)CTGTCTCTTATACACATCTTAATGTCTCCCA(30)GTACTCTGCGTTGATACCACTGCTT -3' 3'- CCCTCTGTAATTCTACACATATTCTCTGTC(NNNNNN)NNNNNN GACAGAGAATATGTGTAGAATTACAGAGGGT(30)CATGAGACGCAACTATGGTGACGAA -5' 3'- GACAGAGAATATGTGTAGAATTACAGAGGG 3'- TGAGACGCAACTATGGTGACGAA -5' (T7-top3-sss) (T7-PCR-R) Needed primers: RT: 5’– AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N –3’ (from clontech kit) 5’– AAGCAGTGGTATCAACGCAGAGT –3’ (T7-PCR-R) (Tm 61.02) 5'- GGGAGACATTAAGATGTGTATAAGAGACAG -3' (T7-top3-sss) (Tm 63.03)
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