IGEM:MIT/2006/Notebook/2006-8-1: Difference between revisions

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==to do (am)==
==to do (am)==
#ask Isadora to help with fax machine
#'''START ASAP!!!''': repeat failed 7/31 digestions '''-done'''
#order new pchA reverse primer (ask Tom for help?)
#order new pchA reverse primer (ask Tom for help?)
#run a gel of BAT2 and THI3 pcr products
#test osmY+E0840(A,B,C) part with plate reader
#test osmY+E0840(A,B,C) part with plate reader
#miniprep/nanodrop 7 overnight liquid cultures
#repeat failed/funky digestions (and run gel)


==to do (pm)==
==to do (pm)==
#gel extract digests products
#nanodrop and run a gel of digest products (NO GEL EXTRACTION)
#8 ligations using 7/31 digested gel extracts
#do ligations using old 7/31 gel extracts
#9 transformations (@2:30 in afternoon)
#do ligations using new 8/1 digest products
#transform everything into new top10 cells


==repeat failed/funky digestions (and then gel extract)==
==Miniprepped LCs==
#R0040: SP
30-ATF1 ABC and osmY-E0840 ABC and R0040
#RBS<sub>30A</sub>-ATF1<sub>mut</sub>: ES
 
#RBS<sub>30B</sub>-ATF1<sub>mut</sub>: ES
==Run Gel of BAT2 and THI3 PCR Products==
#RBS<sub>30C</sub>-ATF1<sub>mut</sub>: ES
Nothing showed (including + control)- so we will PCR BAT2 and THI3 again with 1 &mu;L yeast genomic dna
#RBS<sub>32A</sub>-ATF1<sub>mut</sub>: ES
 
#RBS<sub>32B</sub>-ATF1<sub>mut</sub>: ES
==PCR BAT2 and THI3 (2nd attempt)==
#RBS<sub>32C</sub>-ATF1<sub>mut</sub>: ES
*Used 1&mu;L of Yeast genomic DNA from Samantha...and it worked!!  Tomorrow we will PCR-cleanup, cut, and ligate into pSB1A3.  Then we'll be ready to make the isoamyl alcohol generating device! yay!!!!!! :-D
 
==Fax Dr. Schweizer for pUCP18 etc.==
#original fax number is most likely dead or old
#found 2 new fax numbers and successfully sent a fax to University of Calgary and Dr. Schweizer's updated office fax


==gel extraction pieces from 7/31==
#RBS<sub>30A</sub>-CDS-Term: XP
#RBS<sub>30B</sub>-CDS-Term: XP
#RBS<sub>30C</sub>-CDS-Term: XP
#RBS<sub>32C</sub>-ATF1<sub>mut</sub>: ES
#RBS<sub>32A</sub>-ATF1<sub>mut</sub>: ES
#RBS<sub>30A</sub>-ATF1<sub>mut</sub>: ES
#RBS<sub>30B</sub>-ATF1<sub>mut</sub>: ES
#E0840: XP


==other cut pieces we have ready to use==
==Reminder: Cut Pieces We Have==
#B0015: EX
#B0015: EX
#B0030: SP
#B0030: SP
#B0032: SP
#B0032: SP
#R0040:SP (didn't show with 5&mu;L loaded on wide lane gel, but could still be there)
#R0040:SP (didn't show with 5&mu;L loaded on wide lane gel, but could still be there)
#More on [[../../Index | topical index]]


==ligations (with 7/31 gel extract pieces 1-8 above)==
==Gel extract fragments (7/31)==
#R0040+1->Prom-RBS<sub>30A</sub>-CDS-Term
#RBS<sub>30A</sub>-CDS-Term: XP
#R0040+2->Prom-RBS<sub>30B</sub>-CDS-Term
#RBS<sub>30B</sub>-CDS-Term: XP
#R0040+3->Prom-RBS<sub>30C</sub>-CDS-Term
#RBS<sub>30C</sub>-CDS-Term: XP
#4+Term->RBS<sub>32C</sub>-ATF1<sub>mut</sub>-Term
#RBS<sub>32C</sub>-ATF1mut: ES
#5+Term->RBS<sub>32A</sub>-ATF1<sub>mut</sub>-Term
#RBS<sub>32A</sub>-ATF1mut: ES
#6+Term->RBS<sub>30A</sub>-ATF1<sub>mut</sub>-Term
#RBS<sub>30A</sub>-ATF1mut: ES
#7+Term->RBS<sub>30B</sub>-ATF1<sub>mut</sub>-Term
#RBS<sub>30B</sub>-ATF1mut: ES
#E0840: XP
 
==9 ligations using gel extracts 1-8 (7/31) above==
#R0040+1->Prom-RBS<sub>30A</sub>-CDS-Term
#R0040+2->Prom-RBS<sub>30B</sub>-CDS-Term
#R0040+3->Prom-RBS<sub>30C</sub>-CDS-Term
#4+Term->RBS<sub>32C</sub>-ATF1<sub>mut</sub>-Term
#5+Term->RBS<sub>32A</sub>-ATF1<sub>mut</sub>-Term
#6+Term->RBS<sub>30A</sub>-ATF1<sub>mut</sub>-Term
#7+Term->RBS<sub>30B</sub>-ATF1<sub>mut</sub>-Term
#R0040+8(E0840)->CP-RBS-GFP-Term
#R0040+8(E0840)->CP-RBS-GFP-Term
#osmY+2->osmY-RBS<sub>30B</sub>-CDS-Term
==Take 2 Digestions (same as 7/31)==
#note number system!
#*<1>: RBS<sub>30A</sub>-CDS-Term: XP ***this got really messed up because the number 1 looks like the number 7***
#*<2>: RBS<sub>30B</sub>-CDS-Term: XP
#*<3>: RBS<sub>32A</sub>-CDS-Term: XP
#*<4>: RBS<sub>32C</sub>-CDS-Term: XP
#*<9>: RBS<sub>30A</sub>-ATF1mut: ES
#*<10>: RBS<sub>30B</sub>-ATF1mut: ES
#*<10***>: RBS<sub>30C</sub>-ATF1mut: ES
#*<12>: ATF1mut<sub>B</sub>-Term: XP
#*<13>: ATF1mut<sub>c</sub>-Term: XP
*Order of lanes: 2, 3, 4, RBS<sub>32C</sub>-ATF1mut: ES, 9, 10
[[Image:8-1gel1.jpg]]
*Order of lanes: 10***, 12, 13, 14
[[Image:8-1gel2.jpg]]
==13 ''Take 2'' ligations==
#R0040 + <1> ***BAD...the "1" was really "7"; didn't transform this***
#R0040 + <2>
#R0040 + <3>
#R0040 + <4>
#osmY + <1> ***BAD...the "1" was really "7"; didn't transform this***
#osmY + <4>
#<9> + B0015
#<10> + B0015
#<10***> + B0015
#B0030 + <12>
#B0030 + <13>
#B0032 + <12>
#B0032 + <13>


==transformations (@ 2:30 pm)==
==transformations (@ 5:30 pm)==
#prepare 9 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
#use fresh Top10 cells, pUC19 control, 200 &mu;L
#use fresh Top10 cells, pUC19 control, 200 &mu;L

Latest revision as of 15:30, 1 August 2006

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

PCR BAT2 and THI3 (2nd attempt)

  • Used 1μL of Yeast genomic DNA from Samantha...and it worked!! Tomorrow we will PCR-cleanup, cut, and ligate into pSB1A3. Then we'll be ready to make the isoamyl alcohol generating device! yay!!!!!! :-D

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 [[../../Index | 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


  • Order of lanes: 10***, 12, 13, 14

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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