Protocol

Abbreviation

standard cloning_ANL

Name

standard cloning_ANL

Laboratory name

Argonne National Laboratory

Type

cloning

Description

Day 1: Setup PCR plate, and run it in the thermocycler overnight.


This protocol will produce a well volume of 51 µL:
0.26 mM each primer
1x buffer
0.2 mM each dNTP
1 mM MgSO4
0.016 U/µL KOD polymerase

Notes: - Different gDNAs will be used in different wells, as specified in the plate design.
- gDNA is added in the amount of 8.333 ng/well. Stock gDNA should be 100 ng/µL.
- KOD polymerase should not be thawed. It is a glycerol solution, and you can pipet it at -20°C.
- KOD polymerase is HotStart; inactive until heated. Not necessary to add the polymerase last.

Hazards: Normal Precautions.


1. Begin thawing reagents:
10x KOD buffer
dNTPs
MgSO4
gDNA
primer plates

2. Prepare a master mix according to the PCR Setup spreadsheet. Keep on ice at all times.

3. Divide the master mix into different segments for the different gDNAs, as specified in the spreadsheet.

4. Dispense 40 µL of the correct PCR reaction mix into each well in the PCR plate.

5. Keep PCR plate on ice, and run program “primersetup” to add primers.

6. Centrifuge plate briefly to ensure all liquid is at the bottom of the wells.

7. Run program KOD1 in thermocycler.

Denature 95°C for 3 minutes
Cycle(32x) 95°C for 40 seconds
53°C for 40 seconds
72°C for 1.5 minutes (depends on anticipated product length)
Polish 72°C for 5-10 minutes
Store 4°C overnight.

Runtime: ~2 hours and 30 minutes





Day 2: Verify PCR with Picogreen assay. Purify PCR products.



Hazards: “No data are available addressing the mutagenicity or toxicity of Quant-iT PicoGreen dsDNA reagent. Because this reagent binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.” – PicoGreen manual.
- Try to avoid any contamination of PicoGreen, clean up spills immediately, change gloves if any spills occur. Change gloves after procedure.

Notes: PicoGreen has a high freezing point, and takes a while to thaw.
The data from FluoroStar is delivered by rows, while SG data is usually kept in columns. The data must be re-sorted before pasting into your SG### spreadsheet.

1. At least 20 minutes prior to setup, take the Picogreen dye out of the fridge and allow it to thaw at room temperature, in a dark location.

2. Assemble materials.
PCR frags plate
TE buffer, 25 mL
Black flat bottom 96 well plate
Reservoir with water
Reservoir for diluted PicoGreen

3. Dilute PicoGreen reagent. 25mL of TE are mixed with 125 μl of picogreen dye for every two plates processed. Be sure to use this mixture immediately. It is light sensitive; therefore the longer it sits out in the light, the more your results will be affected.

4. Run Multimek program PicoG1. Setup as below:

5.

6. Setup Fluorostar software.
Open program. Highlight “User” and click “Run”.
Set mode to “Fluorescence”. (box in bottom left of window)

7. Open the top of the FluoroStar machine and rotate the cords so the Fluorescence cords (silver cords) are at the top.

8. Open the Fluoro tray (button at top of window). Place the fluorescent plate. Close the Fluoro tray.

9. Start the program.
Click on the “Traffic Signal” icon.
Select the “Picogreen Assay” program, and press “Ok”.
Name your experiment run.
Click “Start Test Run”.

10. Your results are put into an Excel spreadsheet.
Access this spreadsheet by clicking the “Microsoft Excel” icon in the toolbar.
In the spreadsheet, select your test run and double click.












Overview: Remove primers and dNTPs from the PCR products.
End result is your gDNA and amplified genes, in 150 µL of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) or EB buffer (10 mM Tris·Cl, pH 8.5). Be careful if eluting with TE, the EDTA may inhibit subsequent reactions.

Hazards: Robot; keep clear.

Notes: Watch the robot to make sure that all vacuum steps are performed as expected. It is especially important that the PE buffer is all pulled through before you add EB / TE.

Materials:
PM, PE, and TE buffers
QiaQuick (purple) filter
Deepwell plate
Skirted PCR plate

1. Open the Biomek software, and open program “PCR Purification”. Setup the table as indicated in program.
a. Put your PCR plate in the drilled bottom plate.
b. Be sure to fill the PE reservoir to the brim, the program uses a lot.

2. Run the program. Be sure to turn on vacuum and vacuum switch as instructed, and to blot when instructed. If you have filled the PE reservoir to the brim, there is no need for the mid-procedure refill.

3. Be sure to turn the vacuum pump off.




Day 3: Make your PCR fragments LIC-ready. Perform LIC and transform into BL-21 cells.

LIC Overview: Use the 3’ to 5’ exonuclease activity of T4 polymerase to create overhangs on your PCR frags. Combine 32 μL of PCR pur with 10.8 μL of LIC rxn mix. Final concentrations of LIC reaction in plate:
0.94x T4 polymerase buffer
2.21mM dCTP
4.34mM DTT
2 units T4 DNA polymerase (0.05U/μL)

Notes: This procedure is modified. No mineral oil, no PCR pur dilution.

Hazards: Robot; keep hands clear. DTT is an irritant and health hazard; avoid contact.

Materials: 2 skirted 96 well plates
LIC reaction materials

8. Make LIC Reaction mix as follows for two plates:
1000μL 10x T4 Polymerase buffer
232.5μL 100mM dCTP
45.6μL 1M DTT
1160μL Water
200μL 2.5U/μl T4 DNA Polymerase (Novagen-LIC quality)
2638μL Total

9. Array 10.8 μl of LIC reaction mix into the labeled MJ Thermocycler plates.

10. Multimek – Run program LICxGC. This will transfer 32 µL of PCR pur to the LIC plate, and pipet mix.
11.

12. Foil seal the plates. Incubate at RT for 30 min (up to an hour is permissible).

13. Inactivate enzyme by incubating at 75°C for 20 min.

14. Store at 4°C short term, -20°C for up to several months, or -80°C for even longer periods.

Overview: Anneal the prepared insert into the prepared vector. Then transform and plate.

Hazards: LIC insert solution contains DTT.

Materials: 48-well LB/amp plates
Competent cells, 5 mL / plate (10 mL for 2 plates)
Vector

1. Begin gently thawing comp cells on ice. Label new plate for annealing.

2. Array vector into annealing plate.

3. Add LIC prepared insert, 4 µL per well.

4. Incubate at RT for 10 minutes (5 minutes to 1 hour is ok).

5. Incubate on ice for 2 minutes.

6. Add competent cells, 55 µL per well.

7. Incubate on ice for 5 minutes, to allow cells to recover.

8. Heat shock at 48°C for 45 seconds, to make cells receptive.

9. Incubate on ice for 2 minutes.

10. Add LB medium (SOC is preferable), 60 µL per well (or up to 120 µL)

11. Incubate at 37°C for 30 minutes (up to 1 hour is ok). While they are incubating, prewarm the agar plates.

12. Plate 60 µL. Leave plates uncovered to dry, then invert and incubate at 37°C for ~14 hr. Plates must be dried to separate individual colonies.