Question

Question 19 (1 point) Forward Primer: GTG ATG CGG GTT TGT GCA GAC (5' to 3' on coding strand) Make a reverse primer from the following sequence, also from the coding strand, that is GC clamped and the Tm is within plus or minus 1 degree C (using the rule of thumb for calculating Tm we used in class). Please write the primer in its own 5' to 3' sequence, so we can send it out to be synthesized. ATC GCT GTG CTC CAC GAG ACA GTC CGT TGA (5' to 3' on coding strand) The spacing is just to aid in counting. Please write the primer in its own 5 prime to 3 prime sequence, so we can send it out to be synthesized. OCTC CAC GAG ACA GTC CGT TG OGTG CTC CAC GAG ACA GTC CG O ATC GCT GTG CTC CAC GAG ACA O GAG GTG CTC TGT CAG GCA AC O A CAC GAG GTG CTC TGT CAG GC O TAG CGA CAC GAG GTG CTC TGT OTCA ACG GAC TGT CTC GTG GAG OCG GAC TGT CTC GTG GAG CAC AG OTGT CTC GTG GAG CAC AGC GAT Question 20 (1 point) Forward Primer selection in CAPS: acq gat taa ATA GTG GAG ACA GTC CGC GGG gcgccg gtc. You have been working on making a forward primer on the strand shown above. You wanted it to be 21 bps long (who knows why, but people are weird about this), and you have 21 bps up there in CAPs, so that's good. However, you also want a Tm of exactly 64 degrees. Using the rules for Tm that we learned in class, how can you quickly adjust the selection to get the correct Tm? Using the rules for Tm that we learned in class, how can you quickly adjust the selection to get the correct Tm? Shift the selection 3 bp to the left. Shift the selection 2 bp to the left. O Shift the selection 1 bp to the left Leave it alone; it is fine! Shift the selection 1 bp to the right. Shift the selection 2 bp to the right. Shift the selection 3 bp to the right.

Answer 1

Suestion 19 Forward primer 5 GTG ATG C GG GTT TGT GCA GAC 2' G = NH!!!! - (3+6)* 27+19+3)*4] - 18+ 48 A = ill T = INI = 66°c. Now, coding sequence for reverse primer. Б АТc Qст стестС CAC GAG ACA GTC сQT TGА З 3 TAG CGA CAC GAG GTG CTC TGT GAG GCA ACTS Im of primer of coding sequence. A = NULL = 7 T m = [(25+6)* 2] + [9+8)* 4] T = ITHI = 6 = 26 + 68 Ga nu - 9 3 94°C. C 3 MIN 58S If we remove, 3 codons from 3'end, I'm will decrease by 28°C. [(3+1) *2] + [(2+3)*4] = 8 + 20 = 28 °C so Reverse primer to match Tm of forward primer - 5 TCA ACG GAC TGT CTC GTG GAG 3 Answer

Question 20 acg gat taa ATA GTG GAG ACA GTC CGC - GGG gcg cog gte 31 Im of Forward primer (in caps). 111 A = 14 Tm = [(5+3)* 27 + 16 + 4) * 4] T=1 G - NIL - $6 + 40 c = lill = 56°C. It is A (64 - 56) c = 8°c. 8 required to make not fine it the appropiate Tm esmise o If selection is shift & bp to the left A will increase Im by 2°c. 58°c. • If selection is shift 2 bp to the left AA will increase Tm by 4°C 60°C. I ADT TOTA If selection is shifted 3 bp to the left Tm by 6°c - 62°c • If selection is shifted 1 bp to right Im increases ouring by 4°C 60°c om of A selection is shifted 2 bp to tright I'm increases by 8°c - 64 C. (56°c + 8°c) ASA AT • If selection is shifted 3 bp to right Tnt 12°c 56 + 12°c = 68c