User:Lu Wang/Notebook/Team Allergy/2010/07/08

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Procedures

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

  1. send out plasmids for sequencing with new primers
  2. Miniprep of plasmids containing allergen panel parts
  3. Diagnostic Digest of plasmids
  4. Gradient PCR for PDK intron from pKannibal and pHannibal
  5. Growing more colonies containing pKannibal and pHannibal vectors for more DNA in case if gradient PCR fails to yield enough DNA

Results

Allergen Panel

  • Sent out for sequencing this morning using new primers
  • Minipreps of Allergen Panel
    • Concentrations:

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

  • Diagnostic Digest - Digested with Xba and Pst

Allergen Panel after 40 min.jpg

PDK intron

  • Gradient PCR of PDK intron

PHAN+pKAN PDK PCR.jpg

lanes using 50 & 55 C worked

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)

Name Amount (uL)
10mM dNTPs 1
5x Phusion Buffer 10
Phusion Polymerase .5
pdk 1 uL DNA (12.6 and 16.5 ng)
Fwd/Rev Primer .5 uM each (1uL 50x) at .5 uM
Water 35.5


  • Growing up 5 mL cultures of pHannibal and pKannibal for getting more DNA for pdk pcr
    • Gel purifying/digesting/ligating/transforming pdk intron into e.coli





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