TITLE: Over Expression and Rescue Constructs
AUTHORS: Yaowu Yuan

[STEPS]
?. [PCR Insert and Purify]
Use a high-fidelity enzyme (Phusion) to amplify your insert.  AB1PCR1x2dH2O12.0 µL35x Phusion Buffer4.0 µL4dNTPs (10mM)0.4 µL5DMSO (100%)0.6 µL6F Primer (5µM)*1.0 µL7R Primer (5µM)1.0 µL8Template1.0 µL9Phusion Enzyme**0.2 µL10Total20 µL
AB1PCR1x2dH2O12.0 µL35x Phusion Buffer4.0 µL4dNTPs (10mM)0.4 µL5DMSO (100%)0.6 µL6F Primer (5µM)*1.0 µL7R Primer (5µM)1.0 µL8Template1.0 µL9Phusion Enzyme**0.2 µL10Total20 µL
?. [PCR Program]
PCR Program  for  for  for  for  -  ** for  forever
[*]
*Anneal Temperature depends on primers.**Depends on the length of the fragment (30 sec/1 kb)
?. [PCR Program]
Run PCR product on 1% gel to check for size and a single band.If the PCR product is clean, single band, it can be purified. If there is multiple bands, gel purify the fragment. Elute PCR product in .
[dH2O]
?. [pENTR-D TOPO Cloning Reaction]
Mix and incubate at room temperature for at least , then put on ice. Transform entire reaction into E. coli competent cells and plate all on Kan plates. 
  AB1dH2O1.0 µL2PCR Product*1.0 µL3Salt Solution0.5 µL4Topo Vector0.5 µL5Total3.0 µL
AB1dH2O1.0 µL2PCR Product*1.0 µL3Salt Solution0.5 µL4Topo Vector0.5 µL5Total3.0 µL
?. [Colony PCR to check for the Insert]
Use M13F (or T7F) primer and an insert specific primer (cdsR) to check for the size and direction of the insert.
IMPORTANT! Sometimes the insert could be inserted in the opposite direction.
?. [Making a replica plate and adding template]
Circle and number the colonies you wish to colony PCR (8-16 colonies).
?. [Making a replica plate and adding template]
Get a new selective plate and make a grid and number each cell = Replica Plate
?. [Making a replica plate and adding template]
Using a P20 pipet tip, gently touch the side of a single colony. Gently touch the corresponding grid on the replica plate, then place the pipet tip in the PCR tube containing your master mix.
?. [Making a replica plate and adding template]
Incubate replica plate at  for a few hours or overnight.
?. [Making a replica plate and adding template]
Run PCR product on a gel to check for the presence of an insert in the correct orientation.
?. [Culture two colonies with the correct insert and isolate the plasmid (entry clone)]
In a labeled 15 mL Falcon tube, pipet .
[LB broth]
?. [PCR using M13 F/R primers to amplify fragment for LR reaction]
Necessary since the entry clone and the destination vector both have Kan resistance.Use the Phusion protocol from Step 1  and M13F/R primers to amplify the fragment.Use only 28 cycles since the PCR should be very strong.Gel or PCR purify the fragment.
?. [LR Recombination reaction]
Mix well and incubate at  () for .  AB1dH2O2.5 µL2Entry clone PCR fragment1.0 µL3Destination Vector*0.5 µL4LR Clonase**1.0 µL5Total5.0 µL
AB1dH2O2.5 µL2Entry clone PCR fragment1.0 µL3Destination Vector*0.5 µL4LR Clonase**1.0 µL5Total5.0 µL
?. [Colony PCR using insert specific primers to check for insert]
Use insert specific primers to check for insert or a primer on the vector if available (attR2 for example).
?. [Culture two colonies and isolate the plasmid (final plasmid)]
Culture two colonies in  overnight at  with shaking.
[LB broth + Kan]
?. [Culture two colonies and isolate the plasmid (final plasmid)]
Isolate the plasmid using a Plasmid Mini-Prep Kit. Elute in .
[dH2O]
Elute the plasmid twice (  +  ). Works much better than once ( ).
?. [Sequencing]
Sequence verify the final plasmid.
?. [Transforming One Shot Top10 E. Coli competent cells]
Take out S.O.C from  freezer to thaw.
?. [Transforming One Shot Top10 E. Coli competent cells]
Thaw 1 vial of One Shot cells on ice and aliquot the cells into separate cells into separate PCR tubes, if necessary.
1 vial can do 3 or 4 transformations, but it cannot be refrozen.
?. [Transforming One Shot Top10 E. Coli competent cells]
Add product to be transformed into One Shot cells and mix gently.
DO NOT mix by pipetting up and down.
?. [Transforming One Shot Top10 E. Coli competent cells]
Incubate on ice for .
?. [Transforming One Shot Top10 E. Coli competent cells]
Add  S.O.C to the tube.
250 µl
Add   if the cells were split into 2 or 3 tubes.
?. [Transforming One Shot Top10 E. Coli competent cells]
Immediately transfer the tubes to ice.
?. [Transforming One Shot Top10 E. Coli competent cells]
Heat shock the cells for  at  in the PCR machine.
?. [Transforming One Shot Top10 E. Coli competent cells]
Shake the tube horizontally at  for .
?. [Transforming One Shot Top10 E. Coli competent cells]
Spread - of the transformation on pre-warmed selective plates and incubate overnight at .
50 µl
200 µl
?. [Culture two colonies with the correct insert and isolate the plasmid (entry clone)]
Add of Kan to each tube.
3 µl
?. [Culture two colonies with the correct insert and isolate the plasmid (entry clone)]
Pick a colony with the insert using a P20 tip and eject into falcon tube.
?. [Culture two colonies with the correct insert and isolate the plasmid (entry clone)]
Incubate at  with shaking overnight.
Try to start your cultures in the afternoon and take them out of the shaker in the morning - if left to grow too long, it will start to die.
?. [Culture two colonies with the correct insert and isolate the plasmid (entry clone)]
Isolate the plasmid from the culture using a Plasmid Mini-Prep Kit. Elute plasmid in .
[dH2O]
Elute the plasmid twice (  +  ). Works much better than once ( ).
?. [LR Recombination reaction]
Add  to terminate the reaction and vortex briefly.
[proteinase K]
?. [LR Recombination reaction]
Incubate at  for  .
?. [LR Recombination reaction]
Transform  of the reaction into E.Coli competent cells and plate 20-100 uL on Kan plates.
3 µl
?. [Colony PCR using insert specific primers to check for insert]
Make a replica plate.
?. [Colony PCR using insert specific primers to check for insert]
Run PCR product on a gel to check for the presence of an insert.