LIC-cloning into the pET-NKI LIC vectors
[contributed by Vangelis Christodoulou, NKI]
This protocol is based on the "version 2" LIC system designed by Vangelis.
DAY -20 to -10 - Primer design and ordering Determine the cDNA sequence of the constructs you want to make. Design primers based on this sequence aiming for a 68oC annealing temperature. Add the LIC-specific extensions “cagggacccggt” to the 5’ end of the fwd primer and “cgaggagaagcccggtta” to the 5’ end of the rev primer. For example:
Primers can be ordered from the Sigma Genosys website 0.025 ug quantity and desalting is sufficient. Delivery time can vary considerably allow plenty of time.
DAY0 - Growth induction
From glycerol stocks or a plate inoculate 250 -300 mL LB with 30ug/mL Kanamycin in a 1L flask with either DH5a or Nova Blue cells containing the pET-NKIb 3C/LIC empty plasmid. Grow to saturation overnight at 37oC 140rpm.
DAY1 – Vector cleavage
Perform a Qiagen-Midi or Maxi- prep of the plasmid according to instructions. 1ug of cleaved plasmid will be enough for approximately 20 LIC reactions. At this stage carefully check the size of the plasmid on a 0.8% Agarose gel, the supercoiled plasmid should run just below the MIII Marker 3.5kbp band.
Cleave the vector with the appropriate restriction enzyme (based on the LIC vector you want to use). For example the reaction for 10ug would be something like:
|pET-NKIb 3C/LIC||10ug (e.g. 100uL of 100ng/uL stock)|
|restriction enzyme||10uL of 1U/uL stock|
|MQ H20||Make upto 140uL water|
Make up in a 1.5mL eppendorf and incubate at 37oC for 2-3 hours. Remove a small sample and run on a 0.8% Agarose gel to check cleavage efficiency.
DAY1 – Insert PCR
Setup the PCR of the insert in 500uL thermowell tubes.
|DNA template||X uL||20 ng|
|10x Stratagene Pfu buffer (contains Mg2+)||10 uL||1x buffer|
|10mM dNTPs||2.5 uL||250 uM|
|Fwd primer (100pmol/uL)||0.05-1.0 uL||5-100 pmol|
|Rev primer (100pmol/uL)||0.05-1.0 uL||5-100 pmol|
|Pfu Stratagene polymerase (2.5U/uL)||1.0 uL||2.5U|
|MQ H2O||Upto 100uL|
Run with a cycle such as:
|94oC for 5 minutes|
|94oC for 1 minute }|
|55oC for 1 minute }||Repeat steps 30x|
|72oC for 2.5 minutes }|
|72oC for 10 minutes|
|10oC for ever|
Remove 5 uL of the PCR reaction and mix with 3 uL of 6x Loading dye. Run on a 0.8% Agarose gel and check the size of the bands. Ensure that they are correct (usually by looking for slight movements up/down compared to nearby samples).
Perform a Qiagen PCR-cleanup of the PCR reaction mixtures providing there is a single band only. If two or more bands occur a gel-extraction may be necessary, but if they are clearly distinct these can be more easily separated by performing a larger number of PCR-colony screen
DAY2 – T4 Polymerase treatment
Treat the cut-vector and PCR’d insert with T4 polymerase in 1.5mL eppendorf tubes. The number of pg/pmol = (#of bp)x650. So for a 1000bp PCR-product we have 1000x650 = 650 000 pg/pmol = 650 ng/pmol which means for 0.2pmol of insert we require:
650ng/pmol x 0.2pmol = 130ng
normal yields of Qiagen miniprep are 20ng/uL – 100ng/uL so will require 8uL-1uL of PCR product.
Reagent Add PCR insert 0.2 pmol 10x buffer (NEBuffer #2) 2 uL 25 mM dATP 2 uL T4 polymerase (NEB 3U/uL) 0.8 uL MQ H2O Upto 20 uL
Cut, gel-extracted vector 0.2 pmol
10x buffer (NEBuffer #2) 2 uL
25 mM dTTP 2 uL
T4 polymerase (NEB 3U/uL) 0.8 uL
MQ H2O Upto 20 uL
1) Incubate at 22oC for 30 minutes.
2) Denature the T4 polymerase by then incubating the reaction at 75oC for 10-20 minutes.
3) Spin down the evaporation.
Conjoin the vector and insert
1) Add 1 uL of the T4 treated insert to a 1.5 mL eppendorf tube
2) Add 2 uL of the T4 treated vector (~50ng/uL)
3) Incubate together at 22oC for 5 minutes.
4) Stop the reaction by adding 1 uL of sterile 25 mM EDTA.
Transform the reaction into Novagen NovaBlue’s
1) Add 10-15 uL of Nova Blue competent cells to 2ul of conjoined vector/insert mix.(Store the other 2ul at -20oC). Include a T4 treated vector-only sample to assess background.
2) Incubate on ice for 30 minutes.
3) Heat shock at 42oC for 30-40 seconds.
4) Incubate on ice for 2 minutes.
5) Add 250 uL of SOC media or LB without antibiotic.
6) Allow the cells to recover by incubating at 37oC, 1400rpm.
7) Plate out on the appropriate antibiotic for the vector, leave overnight at 37oC.
DAY3 – Colony screening. This is only one way to screen for positive clones. Other alternatives include a direct expression test or miniprep growth followed by Nco I/ Xho I digest. Restriction enzyme digest is preferable if PCR’s are problamatic, it is a more expensive technique.
If colonies have grown (colonies take closer to 20 hours to grow) perform a PCR-screen using the sequencing primers appropriate for the vector (e.g. T7 promoter/ T7 terminator primers for the pET vector). Setup a Taq sequencing mix such as:
Reagent For 1 reaction For 25 reactions
10 x invitrogen Taq buffer ( - Mg2+) 2 uL 50 uL
50 mM invitrogen MgCl2 1 uL 20 uL
10 mM dNTPs 0.4 uL 10 uL
Fwd primer (10 pmol/uL) 0.3 uL 7.5 uL
Rev primer (10 pmol/uL) 0.3 uL 7.5 uL
Invitrogen Taq polymerase (5U/uL) 0.15 uL 3.75 uL
MQ H2O Upto 20uL 401.25 uL
1) Label 2 or 3 bacterial colonies from each samples plate.
2) Aliquot 20 uL of the master PCR mix into each well of a 96-well thermowell tray.
3) Take a sterile P10 tip and scrape half of each bacterial colony and put this into the 20 uL. Leave the tip in the tray while going onto the next sample.
4) After scraping all colonies use a pipette to mix briefly and seal the reactions.
5) Run using a program such as:
96oC for 10 minutes
96oC for 1 minute |
55oC for 1 minute | Repeat steps 25x
68oC for 1 minutes/kbp |
68oC for 10 minutes
10oC for ever
6) When finished add loading dye to each PCR reaction and run half on a 0.8%-1.0% Agarose gel. Check the size of each band (remember the sequencing primers will add approx 170 extra bp compared to the original reaction). If any samples are missing colonies go back and rescreen.
7) Inoculate overnights from the remaining half-spot of the positive colonies.
DAY4 – Plasmid minipreps.
From the overnights purify plasmids by either using Qiagen mini-preps or in a 96-well block format. Transform into an expression cell-line like BL21 (DE3) or Rosetta (DE3).