User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/14: Difference between revisions

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|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn2
|-
|-
| DNA (500 ng) || 2.7 μL
| DNA (500 ng) || 2.7 μL || 2.7
|-
|-
| 10X buffer (FD clear) || 2.0
| 10X buffer || 2.0 (FD) || 2.0 (tango)
|-
|-
| SacII || 1.0
| SacII || 1.0 || 1.0
|-
|-
| XbaI || 1.0  
| XbaI (FD) || 1.0 || 1.0
|-
|-
| SAP (Roche) || 1.0
| SAP (Roche) || 1.0 || 1.0
|-
|-
| dH<sub>2</sub>O || 12.3
| dH<sub>2</sub>O || 12.3 || 12.3
|-
|-
| &nbsp; || 20.0
| &nbsp; || 20.0 || 20.0
|}
|}


Revision as of 18:37, 14 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> 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.


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/ recovery).
  • Plate on 100 μg/mL amp.


RESULTS (5/15/15)

  • tba


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 = 866, 311, 26
  6. (" 6) mEmerald-H3-23 - X/HindIII = 788, 446, 27
  7. (" 7) mEmerald-H4-23 - X/HindIII = 788, 346, 27
  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. BB 1 = size
2. BB2 = size
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.


Assemblies

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


  • Digest & gel purify vector (Fermentas FD)
  1. pcDNA-dCas9-VP64, S/SacII
Reagent Volumes
DNA (plasmid) up to 25 μL 500 ng 15
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.
    • 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) --- --- ---


  • Digest & dephos insert (PCR clip & clone method)
  1. XbaI-CMV-SpeI-NotI-SacII PCR (clean), X/SacII (see 05/11/15
  • Digest & dephos PCR fragments
    • 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
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL



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