User:Lu Wang/Notebook/Team Allergy/2010/07/06

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Procedures

Today, we isolated plasmid that contained antisense and sense sequences of the allergen panel and introns PME and PAL that we grew last week.

For amiRNA

  1. Nanodrop of miniprepped amiRNA plasmids
  2. Diagnostic digest of amiRNA plasmids
  3. E Gel of digested amiRNA
  4. Send plasmids to Genewiz for sequencing

For ihpRNA

  1. Miniprep of plasmids containing introns for hpRNA
  2. Nanodrop of miniprepped intron plasmids
  3. Miniprep of plasmids containing Allergen Panel sense and antisense
  4. Nanodrop of minipreped allergen plasmids
  5. Diagnostic Digest of introns and allergens
  6. E Gel of digested parts
  7. Extract pKannibal and pHannibal vectors and transform into E. coli
  8. PCR of the PDK intron in pKannibal and pHannibal

Results

For amiRNA

Miniprep of turbo bacteria for plasmids containing sequences for amiRNA

Should obtain plasmids containing the complete sequence for amiRNA. The resulting concentration should be relatively high (<100ng/μL) because all growing bacteria would have the plasmids inside.

Nanodrop of miniprepped amiRNA plasmids

We used 2μL of DNA per nanodrop.

Concentrations in ng/μL
Sample # --> One Two Three
GFP 134.4167.6 207.2
GFP 0.5 163.3 106.9 230.9
LTP 134.0 127.8 165.5
LTP 0.5 158.5 137.2 113.5
Bet 129.4 78.9 124.4

Diagnostic digest of amiRNA plasmids Digested 200~500ng of plasmids with EcoR1 and Spe1. We will then run the digested vectors on an E Gel and look for DNA that is the length of the insert. If such DNA appears on the gel, then we will send it off to be sequenced.

E Gel of digested amiRNA

Image of E Gel plates two and one

We had to run 21 samples, so we used two gels. The orders were:

Gel #1

  1. 1kb Ladder
  2. GFP 1
  3. GFP 2
  4. GFP3
  5. GFP 0.5 1
  6. GFP 0.5 2
  7. GFP 0.5 3
  8. LTP 1
  9. LTP 2
  10. LTP 3
  11. LTP 0.5 1 no visible band for neither plasmid nor insert
  12. LTP 0.5 2

Gel #2

  1. 1kb Ladder
  2. LTP 0.5 3
  3. Bet 1
  4. Bet 2
  5. Bet 3
  6. Neg Control for hpRNA (digested with Xba and Pst, and ligated without new inserts) no visible insert band
  7. Neg Control for intron (digested with Eco and Spe, and ligated without new inserts)
  8. PME Good no visible insert band
  9. PME Dubious no visible insert band
  10. PAL Good colony 1 no visible insert band
  11. PAL Good colony 2
  12. 1kb Ladder

For ihpRNA

Miniprep of plasmids containing introns for hpRNA Nanodrop of miniprepped intron plasmids

We used 2μL of DNA per nanodrop.

Concentrations in ng/μL, cells are filled when applicable
Insert Name Good Dubious Colony 1 Colony 2 Colony 3
PME 85.666.8
PAL 67.7 108.8
Neg Control, Intron 74.0
Neg Contron, hpRNA 63.5 30.1 37.3
  1. Miniprep of plasmids containing Allergen Panel sense and antisense (from overnight cultures)--still need to do preps of cultures grown today
  2. Nanodrop of minipreped allergen plasmids
  3. Diagnostic Digest of introns and allergens
  4. E Gel

Gel #1

Lane 1: 1kb Ladder

Lanes 2-4: Ger Sense

Lanes 5-7: Ger Antisense

Lanes 8-10: LTP Sense

Lanes 11-13: LTP Antisense

Lanes 14-16: Bet 2 Sense

Lanes 17-19: Bet 2 Antisense

Lanes 20-22: Bet 1 Sense

Lanes 23-25: Bet 1 Antisense


phannibal and pkannibal Extracted vectors from a piece of paper per instructions. Transformed vectors into E. coli. Miniprep and PCR to extract PDK intron.

  • pcr to get out pdk intron
  • Insert content here...



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