User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/14

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Karmella's BioBrick Cloning Main project page
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05/14/15

  • Ryan - minipreps & digests
  • Rene - PCR cloning trial 2
  • Cas-tone - assembly stage 1



Minipreps

  • Check with E/KpnI digests
  1. pAub-AubR/MRV
  2. pBja-BjaR/MRV
  3. pBra-BraR/MRV
  4. pRpa-RpaR/MRV


Reagent Volume Expected:
1,2. pAub/AubR/MRV = 4240
3,4. pBja/BjaR/MRV = 4099
5,6. pBra/BraR/MRV = 4191
7,8. pRpa/RpaR/MRV = 4116
9. Empty MRV = 2230, 971

np pro. Reg/MRV = ~4000, 971
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
KpnI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~10 min.


CONCLUSIONS

  • Still getting either empty Regulator-MRV vector or an uninterpretable 2 kb band!
  • Measure concentrations, hand off to Ryan for sequencing across promoter insertion site



PCR cloning trial 2

  • Use the CloneJET PCR Cloning Kit - MAN0012707 CloneJET PCR Cloning (manual)
    • pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products (Phusion PCR)
    • All common laboratory E. coli strains can be directly transformed with the ligation product
    • Re-circularized vector expresses toxic enzyme, no blue/white screening needed
    • Optimal insert : vector (0.05 pmol ends) ratio is 3:1; Calculation: length of insert DNA x 0.05 = ng insert to use


  • Ligations & transformations - CloneJET PCR Cloning Kit
  1. Luc14 + CRISPR g034; size = 630 (2.0 μL insert, from 5/13/15)
  2. Gal4EED/luc + CRISPR g034 = 630 (2.0 μL insert, from 5/13/15)
  3. Luc14 + CRISPR g034; size = 630 (4.0 μL insert)
  4. Gal4EED/luc + CRISPR g034 = 630 (4.0 μL insert)


Reagent Volume
2x reaction buffer 10.0
PCR product (>31.5 ng) 4.0
pJET1.2/blunt vector 1.0
T4 ligase 1.0
dH2O 4.0
  20.0 μL
  • Incubate the ligation mixture at room temperature (22°C) for 5 min.
  • Transform 50 μL DH5α-turbo with 10 μL ligation reaction. Follow long protocol (42°C heat shock/ SOC recovery).
  • Pellet at top speed for 3 min.
  • Resuspend in 100 μL of 100 μg/mL amp LB. Plate on 100 μg/mL amp.


RESULTS (5/15/15)

  • Fail - Even fewer colonies than trial 1! Long transformation protocol does not appear to improve the outcome.
  • Stick with the quick transformation.
  • Get more PCR insert DNA from Rene. Will have to do several ligation/transformations to get a good sample size.
  • Start isolating clones to build up library for PCR and melt analysis.



Cas-tone Project

  • STAGE 1 - pcDNA-dCas9-VP64 vector re-design
    • Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo
    • Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII
  • STAGE 2 - Histone parts
    • PCR-clone histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will introduce unnatural amino acids onto conserved histone N-terminal tail
    • Order primers
  • STAGE 3 - Cas-fusion
    • Design primers for H2A, H2B, H3, H4, and tails
    • Insert Kozak-histone parts (X/N) into dCas9 (S/N)
  • STAGE 4 - gRNA
    • Put pre-existing gRNA (from luc experiment) into pSPgRNA


Minipreps & Digests

  1. (prep 2) pcDNA-dCas9-VP64 - X/HindIII = 6450, 2345, 1017
  2. (" 1) pSPgRNA - BbsI = 3201, 22
    No XbaI or HindIII sites
  3. (" 3) pHR-SFFV-dCas9-BFP-KRAB - X/HindIII = 4955, 3127, 2606, 840, 587, 563, 556, 535, 255, 107, 24, 12
  4. (" 4) pMSCV-LTR-dCas9-VP64-BFP - X/HindIII = 5517, 3638, 927, 733, 554
    Low copy, use 5.0 uL
  5. (" 5) mEmerald-H2A-10 - X/HindIII = >337, 866
    Prediction based on annotated insert. "mEmerald-C1" vector sequence not available.
  6. (" 6) mEmerald-H3-23 - X/HindIII = >507, 754
    Prediction based on annotated insert. "mEmerald-C1" vector sequence not available.
  7. (" 7) mEmerald-H4-23 - X/HindIII = >373, 788
    Prediction based on annotated insert. "mEmerald-C1" vector sequence not available.
  8. (from Vi's project) H2B-GFP (pEGFP-N1) - X/HindIII = 3944, 1163


Reagent Rxns 1,3,5-8 Rxn 2 Rxn 4 Expected:
1. pcDNA-dCas9-VP64 = 6450, 2345, 1017
2. pSPgRNA = 3201, 22
3.pHR-SFFV-dCas9-BFP-KRAB - X/HindIII = 4955, 3127, 2606, 840, 587, 563, 556, 535, 255, 107, 24, 12
4. pMSCV-LTR-dCas9-VP64-BFP - X/HindIII = 5517, 3638, 927, 733, 554
5. H2A = >337, 866
6. H3 = >507, 754
7. H4 = >373, 788
8. H2B = 3944, 1163
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 2.0 5.0
10X buffer (FD) 1.5 1.5 1.5
XbaI/BbsI (FD) 1.0 1.0 1.0
HindIII (NEB HF) 1.0 --- 1.0
dH2O 9.5 10.5 4.5
  15 μL    

--> 37°C/ ~10 min.


CONCLUSIONS

  • NEB HindIII HF works well in FD buffer (based on lanes 1 and 3)
  1. Good, expected = observed
  2. Good, expected = observed
  3. Good, expected = observed
  4. Uncertain, expected ≠ observed
  5. Uncertain, expected ≠ observed
  6. Uncertain, expected ≠ observed
  7. Uncertain, expected ≠ observed
  8. Uncertain, expected ≠ observed. Look like it was cut once. Band length matches plasmid size (5107).
  • XbaI blocked by methylation in 5-8?
    • Digest the histone plasmids with different enzymes (tomorrow).


Assemblies

  1. CMV-dCas-VP64_pcDNA: CMV/(X/SacII)/588 + pcDNA-dCas9-VP64/(S/SacII)/9056


  • Digest & gel purify vector
  1. pcDNA-dCas9-VP64, S/SacII - FD buffer
  2. pcDNA-dCas9-VP64, S/SacII - Tango buffer
Reagent Rxn1 Rxn2 Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 15.0 15.0
10x buffer 3.0 (FD) 3.0 (Tango)
XbaI 1.0 1.0
SacII 1.0 1.0
dH2O 10.0 10.0
  30 μL 30 μL

--> 37°C/ ~30 min.

CONCLUSIONS

  • Note: SacII is not a FD enzyme. Tango buffer was supplied with it.
  • Looks like both buffers work fine for both enzymes!


  • Gel purify: Sigma GenElute; elute & back elute w/ 25 μL elution sln.
    • Measure conc.
Sample OD260 260/280 ng/μL
1. pcDNA-dCas9-VP64 (S/SacII) 0.114 1.859 113.5
2. pcDNA-dCas9-VP64 (S/SacII) 0.115 1.885 114.7


  • Digest & dephos insert (PCR clip & clone method)
    • XbaI-CMV-SpeI-NotI-SacII PCR (clean), X/SacII (see 05/11/15 for concentration
    • Final [DNA] = 25 ng/μL
Reagent Rxn1 Rxn2
DNA (500 ng) 2.7 μL 2.7
10X buffer 2.0 (FD) 2.0 (tango)
SacII 1.0 1.0
XbaI (FD) 1.0 1.0
SAP (Roche) 1.0 1.0
dH2O 12.3 12.3
  20.0 20.0
  • LabNet OptiMax Thermocycler: AnOlig shrt
    • 37°C, 10 min
    • 95°C, 5 min
    • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ...25°C/1 min]
    • 25°C, ∞


  • Ligations
    • 588 bp insert / 9056 bp vector * 2 * 50 = 6.5 ng insert
  1. CMV PCR (X/SacII dp FD) + pcDNA-dCas9-VP64 (S/SacII)
  2. CMV PCR (X/SacII dp Tango) + pcDNA-dCas9-VP64 (S/SacII)
  3. pcDNA-dCas9-VP64 (S/SacII)
Reagent Rxn1,2 Rxn2
Insert DNA 0.5 ---
Vector DNA 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 3.0 3.5
  10 μL 10 μL


RESULTS

  • Ligations plates, ~40 colonies
  • Neg ctrl plate, ~25 colonies
  • Pick 2 colonies from each ligation plate for 5 mL cultures



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