User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21

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04/21/15

  • Ryan - regulator assemblies
  • Rene - check gRNA assemblies; digests & sequencing
  • LCR Development - Phusion PCR



Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • DONE - Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


  • Clean up PCR products
    • Zymo clean and concentrator
    • Elute w/ 25 μL dH2O


  • Assemblies
  1. AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
  2. BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
  3. BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
  4. RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201


  • Digests
    • E/X
  1. AubR, 851 bp
  2. BjaR, 776 bp
  3. BraR, 776 bp
  4. RpaR, 779 bp
  5. Modular Receiver Vector (MRV)
Reagent Rxn 1-4 Rxn 5
DNA 10.0 5.0
10X buffer 3.0 3.0
EcoRI 1.0 1.0
XbaI 1.0 1.0
dH2O 15.0 20.0
  30.0 30.0


  • Column clean-up
    • Zymo clean and concentrator


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. AubR-PCR (E/X) -0.005 5.875 -5.001
2. BjaR-PCR (E/X) -0.005 9 -4.788
3. BraR-PCR (E/X) -0.005 7.143 -5.291
4. RpaR-PCR (E/X) -0.003 29 -3.052
5. MRV (E/X) 0.007 1.388 7.11
  • Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.


  • Ligations
  Rxn 1-4 Neg
Insert DNA 3.0 ---
Vector DNA (25 ng) 3.5 3.5
2x lgn buf (Roche) 7.5 7.5
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.0
  15 μL 15 μL
  • Transform 50 uL DH5α-turbo
  • Plate on 100 μg/mL amp

RESULTS (4/22/15)

  1. AubR/MRV: 11 colonies
  2. BjaR/MRV: 11 colonies
  3. BraR/MRV: 6 colonies
  4. RpaR/MRV: 7 colonies
  5. MRV (neg): 3 colonies
  • Success! Pick 2 colonies from plates 1-4 for streak plate and liquid cultures (5 mL each)



Rene - check gRNA assemblies

  • Minipreps from 4/20/15
    • Note: ran out of time last week to grow up cultures. Stored at 4C until this week, grew for ~5 hours and then miniprepped w/ Sigma GenElute kit (elution vol. = 75 μL)
  • Check w/ BpiI/XbaI
  1. gRNA51, 1
  2. gRNA51, 2
  3. gRNA52, 1
  4. gRNA52, 2
  5. gRNA53, 1
  6. gRNA53, 2 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
  7. gRNA54 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
  8. gRNA55, 1
  9. gRNA55, 2
  10. gRNA56, 1
  11. gRNA56, 2
  12. pX330 (ctrl)


Reagent Rxn 1-11 Mix(x13) Expected
pX330 = ~8335, 195
gRNA+pX330 = 8530
File:KAH042115 gel1.jpg
15 μL/lane; 1% agarose; Ladder
DNA 3.0 ---
10X buffer 1.5 19.5
BpiI 0.5 6.5
XbaI 0.5 6.5
dH2O 9.5 123.5
  15.0


  • Conclusion
    • Gel inconclusive, expected sizes not seen for pX330 control
    • Proceed with sequencing
  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. gRNA51-1 0.233 1.928 232.82
2. gRNA51-2 0.112 1.901 112.471
3. gRNA52-1 0.203 1.901 203.39
4. gRNA52-2 0.237 1.923 236.79
5. gRNA53-1 0.212 1.903 212.34
6. gRNA55-1 0.233 1.906 233.19
7. gRNA55-2 0.242 1.896 242.43
8. gRNA55-1 0.217 1.895 217.49
9. gRNA55-2 0.264 1.896 264.27


Sequencing list:

  1. g51-1, f (P139) - Confirmed
  2. g51-1, r (P140) - low Phred 20 score
  3. g51-2, f - Confirmed
  4. g51-2, r - low Phred 20 score
  5. g52-1, f - Confirmed
  6. g52-1, r - low Phred 20 score
  7. g52-2, f - Confirmed
  8. g52-2, r - low Phred 20 score
  9. g53-1, f - Confirmed
  10. g53-1, r - low Phred 20 score
  11. g55-1, f - Confirmed
  12. g55-1, r - low Phred 20 score
  13. g55-2, f - Confirmed
  14. g55-2, r - low Phred 20 score
  15. g56-1, f - Confirmed
  16. g56-1, r - low Phred 20 score
  17. g56-2, f - questionable sequence across gRNA, discard
  18. g56-2, r - low Phred 20 score


  • Cultures for new minipreps
    • 6 & 7 were accidentally mixed - discard these
    • Set up 5 mL cultures for gRNA53-2 and gRNA54



LCR Development

  • Final round of Phusion PCR reactions

Phusion PCR:

  1. MV10 + pol
  2. MV10 + pol
  3. MV10 no pol (neg ctrl to see how much of band is template)
Reagent Rxn1,2 Rxn3 Expected:
1,2. MV10 = 5191
3. No product
Hover name
5 μL/lane; 1% agarose; Ladder
DNA(clean linear) 1.0 μL 1.0 μL
10 μM F primer 1.0 1.0
10 μM R primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion Pol. 0.5 ---
5X HF buffer 10.0 10.0
dH2O 35.5 36.0
  50.0 50.0

Program: Phusion (block A)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 65°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞


Conclusion

  • Lane 3 shows product. Must have accidentally added Phusion pol.
  • Desired band not any brighter than reaction done at 60°C (see 04/17/15).
  • Use reactions from 4/17/15 instead of these for LCR.


Phusion PCR:

  1. Gal4DB-mCh - plasmid
  2. "
  3. ATF2 - plasmid template
  4. "
  5. ATF2 - 1:10 iPSC cDNA template
  6. "
Reagent Rxn1-4 Rxn5,6 Expected:
5,6. Gal4DB-mCh = 1170
7-10. ATF2 = 906
Hover name
5 μL/lane; 1% agarose; Ladder
DNA(clean linear) 0.5 μL 2.0 μL
10 μM F primer 1.0 1.0
10 μM R primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion Pol. 0.5 0.5
5X HF buffer 10.0 10.0
dH2O 36.0 34.5
  50.0 50.0

Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 60 sec]
  • 72°C, 10 min
  • 4°C ∞


Conclusion

  • Gal4DB-mCh - much better than previous reactions. Purify these (combined) and use for LCR
  • ATF2 - rxns w/ plasmid template look awesome! cDNA templates give a spurious band. Use plasmid PCR's (combined, purified) for LCR.