User:Lu Wang/Notebook/Team Allergy/2010/06/28
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ProceduresToday, our objective is to finish the construct for amiRNA so that it will be ready for insertion into V0120. We will run a series of PCR reactions to replace the existing interference sequence with our own sequence using a series of primers. Then, we will sew the small pieces of DNA together in another PCR reaction. Creating Parts of amiRNA
Ligating Parts Together
ResultsCreation of Parts 1,2,3 (Primers A&4; 3&2; 1&B)Annealing Temp: (LTP & Bet)60C (GFP)69C Extension Temp: 15 sec Total of nine reactions: three pieces for each GFP, Betv1, and LTP. 9 (3*3) reactions (GFP; Bet; LTP)
Nanodrop Concentrations: LTP 1: 5.4 ng/uL 2: 5.5 ng/uL 3: 1.2 ng/uL GFP 1: 17 ng/uL 2: 6.6 ng/uL 3: 15.1 ng/uL Bet 1:7.4 ng/uL 2: 29.6 ng/uL 3: 34.3 ng/uL
PCRs A& B worked: 1 (ladder); 2(LTP 1+2); 3 (Bet 1+2); 4(GFP 1+2); 5(Ladder); 6(LTP 1,2,3); 7(Be 1,2,3); 8(GFP 1,2,3); 9(LTP 1,2,3(using .5ul)); 10(GFP 1,2,3 (using .5 uL)) Lanes 2-4 should be ~a little less than 450 bp and 6-10 should be ~450bp Ligation of PartsA: Mix of Parts 1,2,3 w/ primers A&B (Three in one PCR, or simultaneous PCR) Annealing Temp: 60C Extension Time: 15 sec 3 reactions (GFP; Bet; LTP)
Concentrations: GFP 47.3 ng/uL; LTP 79.7 ng/uL; Bet 80.4 ng/uL 2 reactions (GFP, LTP)
Concentrations: GFP 51.5 ng/uL; LTP 67.3 ng/uL B: Step 1: Mix of Parts 1&2 w/ Primers A&2, Step one of two piece PCR 3 reactions (GFP; Bet; LTP)
Concentrations: GFP 45.9 ng/uL; LTP 17.8 ng/uL; Bet 24.2 ng/uL Bands actually not what we are looking for (should be a little larger) Step Two of Two Piece PCR Sewing together parts from step one with part number three of each allergen with primers A and B. Result was successful -- had bands that were ~500bps in length.
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