User:Cassandra M Barrett/Notebook/Open Chromatin/2015/11/11

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LCR pJET reattemped

Purpose: to verify that LCR can be successfully performed on two linear parts. Parts will be fused using LCR and ligated into a pJET vector. Ligation product will be transformed into 'E. coli' and colony PCR (and possibly sequencing) will be performed to confirm properly assembled insert (pJET has sequencing primers already designed for just outside of insertion point)

Methods:

Before beginning, two samples of Gal4DB_mCh and ATF2 previously amplified on 10/4/15 and 9/18/15 were run on a gel to confirm their identities. Samples from 9/18/15 were used and diluted to 30nM.

Perform LCR on Gal4DB_mCh and ATF2 parts. Following reactions are from Ben Nyer's work on 11/9/15


PNK Reaction

Use polynucleotide kinase (PNK) to add 5' phosphate groups to DNA fragments.

  • Gal4DB_mCh (30nM) 2uL
  • ATF2 (30nM) 2uL
  • 10x T4 Ligation buf (NEB) 1.0uL
  • T4 PNK 1uL
  • Water 4uL

Incubate at 37°C/ 30 min. Heat-inactivate PNK at 65°C/ 20 min.


Ligase Cycling Reaction

In a PCR tube, set up the following reaction.

Reagent Volume
PNK DNA 10 μL
dsDNA Oligo Bridge 3.0
10X Ampligase Buffer 3.0
DMSO 2.4
Betaine (5 M) 2.7
Ampligase 1.0
dH2O 7.9 μL
  30.0 μL

Thermal cycler program:

  1. 2 min at 94 °C
  2. 10 s at 94 °C
  3. 30 s at 55 °C
  4. 60 s at 66 °C
  5. Repeat steps 2-5 x50 times
  6. hold at 4 °C
  • 4C hold

pJET Cloning

    • pJET1.2/blunt vector is 5'-phosphorylated and accepts blunt products
    • 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


Reagent Volume
2x Roche lig buf 5.0
LCR product 3.0
pJET1.2/blunt vector 1.0
NEB T4 ligase 1.0
  10.0 μL
  • Set up two ligations, one with LCR product and the other with water as a control
  • Incubate the ligation mixtures at room temperature (22°C) for 5 min.
  • Transform 50 μL DH5α-turbo with 10 μL ligation reactions.

Follow standard DH5alpha transformation protocol:

  • Add 50uL of DH5alpha turbo cells to each tube with appropriate sample (10uL LCR pJET lig, 10uL water ligation control, 10 of water as -control, and 2uL of MV10 as +control). 4 reactions total
  • Incubate on ice for 30min
  • Heat shock at 42C for 35sec
  • Incubate on ice for 2min
  • Add 750uL of SOC media (RT) to each tube
  • Shake 37C for 1hr
  • Plate on LB+AMP (Spin down, remove 200uL of supernatant, resuspend and plate 100uL)
  • Incubate overnight at 37C



Results 11/12/15

Colony Counts

  • negative control: 1 mucoid colony
  • positive control (MV10): lawn
  • pJET H2O ligation: 1 fuzzy colony, 1 mucoid colony
  • pJET LCR ligation: 5 colonies

All five colonies from the pJET LCR plate were used to inoculate 5 separate tubes of LB+AMP broth for a plasmid miniprep. Colonies growing on the pJET H20 plate and negative control could be due to the low level of AMP resistance in the DH5 alpha turbo strain used in the lab. It is possible also that a mutation/insertion occurred in the pJET vector, shifting the kill gene out of frame.


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