IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/28: Difference between revisions
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==Team Allergy== | ==Team Allergy== | ||
Today, 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. | |||
===Procedures=== | |||
Annealing Temp: 60C | Creating Parts of amiRNA | ||
Extension | #PCR reactions of allergen panel with primers (A,IV), (III,II), and (I,B) | ||
#Gel Electrophoresis to find parts | |||
#Gel Purification for Parts | |||
Ligating Parts Together | |||
#Three in one sewing PCR | |||
#Two in one sewing PCR, followed by another two in one PCR | |||
===Results=== | |||
====Creation 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. | |||
''3 reactions (GFP; Bet; LTP)'' | ''9 (3*3) reactions (GFP; Bet; LTP)'' | ||
{| class="wikitable" border ="1px solid black" | {| class="wikitable" border ="1px solid black" | ||
|- | |- | ||
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|.5 | |.5 | ||
|- | |- | ||
|DNA ( | |DNA (RS300) | ||
| | |~10ng | ||
|- | |- | ||
| | |Reaction 1(A&4) Reaction 2(3&2) Reaction 3(1&B) primer mix (1x) | ||
|2.5 | |2.5 | ||
|- | |- | ||
Line 40: | Line 54: | ||
|} | |} | ||
''Concentrations'': GFP | '''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 | |||
'''Gel Electrophoresis''' | |||
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 | |||
[[Image:Amirnaassembly.jpg|400px]] | |||
====Ligation of Parts==== | |||
'''A: 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)'' | ||
{| class="wikitable" border ="1px solid black" | {| class="wikitable" border ="1px solid black" | ||
|- | |- | ||
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|- | |- | ||
|DNA (Parts "1" "2" "3") | |DNA (Parts "1" "2" "3") | ||
|1( | |1(parts 1&2); .5 (part 3)= 2.5 total | ||
|- | |- | ||
|AB primer mix (1x) | |AB primer mix (1x) | ||
Line 68: | Line 104: | ||
|} | |} | ||
''Concentrations'': GFP | ''Concentrations'': GFP 47.3 ng/uL; LTP 79.7 ng/uL; Bet 80.4 ng/uL | ||
'' | ''2 reactions (GFP, LTP)'' | ||
{| class="wikitable" border ="1px solid black" | {| class="wikitable" border ="1px solid black" | ||
|- | |- | ||
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|.5 | |.5 | ||
|- | |- | ||
|DNA (Parts "1" "2") | |DNA (Parts "1" "2" "3") | ||
|1(each)= | |1(each)= 3 total | ||
|- | |- | ||
| | |AB primer mix (1x) | ||
|2.5 | |2.5 | ||
|- | |- | ||
|Water | |Water | ||
|35 | |35 | ||
|- | |- | ||
|} | |} | ||
''Concentrations'': GFP | ''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)'' | ||
{| class="wikitable" border ="1px solid black" | {| class="wikitable" border ="1px solid black" | ||
|- | |- | ||
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|.5 | |.5 | ||
|- | |- | ||
|DNA ( | |DNA (Parts "1" "2") | ||
| | |1(each)= 2 total | ||
|- | |- | ||
| | |A2 primer mix (1x) | ||
|2.5 | |2.5 | ||
|- | |- | ||
|Water | |Water | ||
|35 | |35.5 | ||
|- | |- | ||
|} | |} | ||
''Concentrations | ''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) | |||
==Team Flavour== | ==Team Flavour== |
Revision as of 12:12, 9 July 2010
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Team AllergyToday, 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. ProceduresCreating 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) Team FlavourMiniprep of Miraculin and Brazzein Constructs
DTL (Digestion, Ligation, Transformation) of The Big Three (pENTCUP2, NOSt, NOSt + STOP)
Team Genetic FenceB21 InnoculationInoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone. PCR of Gal4DBD and Barnase, attempt 2Ran 3 tubes of each for a total of 6. 7x Mastermix:
Each tube contained:
Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program Gel of Gal4 DBD and Barnase from second PCR attemptRan 1.2% E-gel of the 6 PCR product tubes.
QIAQuick Purification of PCR ProductBecause colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.
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