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

From OpenWetWare
Jump to navigationJump to search
Line 47: Line 47:


----
----
'''Rene - '''
'''PCR cloning trial 2'''
 
* Use the CloneJET PCR Cloning Kit - [https://tools.lifetechnologies.com/content/sfs/manuals/MAN0012707_CloneJET_PCR_Cloning_UG.pdf 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 - CloneJET PCR Cloning Kit
# Luc14 + CRISPR g034; size = 630
# Gal4EED/luc + CRISPR g034 = 630
 
 
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| 2x reaction buffer || 10.0
|-
| PCR product (>31.5 ng) || 4.0
|-
| pJET1.2/blunt vector || 1.0
|-
| T4 ligase || 1.0
|-
| dH<sub>2</sub>O || 4.0
|-
| &nbsp; || 20.0 μL
|}
 
* Incubate the ligation mixture at room temperature (22°C) for 5 min.
* Transform DH5α-turbo with 10 μL ligation reaction. Follow long protocol (heat shock/ recovery.
* Plate on 100 μg/mL amp.
 
 
RESULTS (5/15/15)
* tba




Revision as of 16:48, 14 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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 - CloneJET PCR Cloning Kit
  1. Luc14 + CRISPR g034; size = 630
  2. Gal4EED/luc + CRISPR g034 = 630


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 DH5α-turbo with 10 μL ligation reaction. Follow long protocol (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


  • 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



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/05/14&oldid=884219"