IGEM:MIT/2006/Notebook/2006-8-1

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(13 ''Take 2'' ligations)
(transformations (@ 2:30 pm))
Line 83: Line 83:
#B0032 + <13>
#B0032 + <13>
-
==transformations (@ 2:30 pm)==
+
==transformations (@ 5:30 pm)==
#prepare 23 plates, review the resistance of backbones
#prepare 23 plates, review the resistance of backbones
#*everything in B0015 backbone is Amp/Kan!! rest are just Amp
#*everything in B0015 backbone is Amp/Kan!! rest are just Amp
#use fresh Top10 cells, pUC19 control, 200 &mu;L
#use fresh Top10 cells, pUC19 control, 200 &mu;L

Revision as of 16:56, 1 August 2006

Contents

to do (am)

  1. START ASAP!!!: repeat failed 7/31 digestions -done
  2. order new pchA reverse primer (ask Tom for help?)
  3. test osmY+E0840(A,B,C) part with plate reader

to do (pm)

  1. nanodrop and run a gel of digest products (NO GEL EXTRACTION)
  2. do ligations using old 7/31 gel extracts
  3. do ligations using new 8/1 digest products
  4. transform everything into new top10 cells

Miniprepped LCs

30-ATF1 ABC and osmY-E0840 ABC and R0040

Run Gel of BAT2 and THI3 PCR Products

Nothing showed (including + control)- so we will PCR BAT2 and THI3 again with 1 μL yeast genomic dna

Fax Dr. Schweizer for pUCP18 etc.

  1. original fax number is most likely dead or old
  2. found 2 new fax numbers and successfully sent a fax to University of Calgary and Dr. Schweizer's updated office fax


Reminder: Cut Pieces We Have

  1. B0015: EX
  2. B0030: SP
  3. B0032: SP
  4. R0040:SP (didn't show with 5μL loaded on wide lane gel, but could still be there)
  5. More on topical index

Gel extract fragments (7/31)

  1. RBS30A-CDS-Term: XP
  2. RBS30B-CDS-Term: XP
  3. RBS30C-CDS-Term: XP
  4. RBS32C-ATF1mut: ES
  5. RBS32A-ATF1mut: ES
  6. RBS30A-ATF1mut: ES
  7. RBS30B-ATF1mut: ES
  8. E0840: XP

9 ligations using gel extracts 1-8 (7/31) above

  1. R0040+1->Prom-RBS30A-CDS-Term
  2. R0040+2->Prom-RBS30B-CDS-Term
  3. R0040+3->Prom-RBS30C-CDS-Term
  4. 4+Term->RBS32C-ATF1mut-Term
  5. 5+Term->RBS32A-ATF1mut-Term
  6. 6+Term->RBS30A-ATF1mut-Term
  7. 7+Term->RBS30B-ATF1mut-Term
  8. R0040+8(E0840)->CP-RBS-GFP-Term
  9. osmY+2->osmY-RBS30B-CDS-Term

Take 2 Digestions (same as 7/31)

  1. note number system!
    • <1>: RBS30A-CDS-Term: XP ***this got really messed up because the number 1 looks like the number 7***
    • <2>: RBS30B-CDS-Term: XP
    • <3>: RBS32A-CDS-Term: XP
    • <4>: RBS32C-CDS-Term: XP
    • <9>: RBS30A-ATF1mut: ES
    • <10>: RBS30B-ATF1mut: ES
    • <10***>: RBS30C-ATF1mut: ES
    • <12>: ATF1mutB-Term: XP
    • <13>: ATF1mutc-Term: XP
  • Order of lanes: 2, 3, 4, RBS32C-ATF1mut: ES, 9, 10

Image:8-1gel1.jpg


  • Order of lanes: 10***, 12, 13, 14

Image:8-1gel2.jpg

13 Take 2 ligations

  1. R0040 + <1> ***BAD...the "1" was really "7"; didn't transform this***
  2. R0040 + <2>
  3. R0040 + <3>
  4. R0040 + <4>
  5. osmY + <1> ***BAD...the "1" was really "7"; didn't transform this***
  6. osmY + <4>
  7. <9> + B0015
  8. <10> + B0015
  9. <10***> + B0015
  10. B0030 + <12>
  11. B0030 + <13>
  12. B0032 + <12>
  13. B0032 + <13>

transformations (@ 5:30 pm)

  1. prepare 23 plates, review the resistance of backbones
    • everything in B0015 backbone is Amp/Kan!! rest are just Amp
  2. use fresh Top10 cells, pUC19 control, 200 μL
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