User:Lu Wang/Notebook/Team Allergy/2010/07/08
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We finished our amiRNA constructs in RS300 and allergen panel parts in V0120 yesterday. Our sequencing results from Genewiz yesterday were bad because we had bad primers. Yesterday's PCR reaction of the PDK intron from pKannibal and pHannibal did not yield products, so we will run them again today.
Today, we will send them in for sequencing again and extract plasmids containing allergen panel parts from E. coli. We will also run a gradient PCR for PDK intron from pKannibal and pHannibal.
For Allergen Panel
LTP 1,2,3: 265.5 ng/uL, 507.1 ng/uL, 275.5 ng/uL;
GerA 1,2,3,4: 283.1 ng/uL, 185.9 ng/uL, 182.4 ng/uL, 314.7 ng/uL;
Bet 2A 1,2,3,4: 246.6 ng/uL, 251.4ng/uL, 550.3 ng/uL, 235.8 ng/uL;
Bet 2S 1,2,3,4,5,6: 613 ng/uL, 296.8 ng/uL, 333.1 ng/uL, 467.3 ng/uL, 230.3 ng/uL, 129.4 ng/uL;
Bet 1A 1,2,3,4,5: 505.8 ng/uL, 632.1 ng/uL, 561.7 ng/uL, 594.4 ng/uL, 609.2 ng/uL
Gel of gradient PCR of the 741bp PDK intron out of pHANNIBAL and pKANNIBAL. Lanes are (left to right, temperatures are annealing temperatures used in the gradient PCR) 1kb+ ladder, pHAN 50ºC, pKAN 50ºC, pHAN 55ºC, pKAN 55ºC, pHAN 60ºC, pKAN 60ºC, pHAN 65ºC, pKAN 65ºC.
It appears that pHANNIBAL worked at 50, 55ºC but pKANNIBAL didn't.
Concentration: hannibal pdk 12.7 ng/uL kannibal pdk: 15.6 ng/uL
Did a PCR of the PCR product
Annealing Temps: hannibal pdk (50 C); kannibal pdk (55 C)
Extension Time: 30 sec
12 reactions (6 hannibal pdk; 6 kannibal pdk)