TITLE: Barcoded vector cloning
AUTHORS: Raining Wang, Melinda Wheelock

[DESCRIPTION]
This is the protocol for inserting barcodes to the N- and C- vector.

[BEFORE_START]
Ensure there are enough maxi-prep kits available to use.
Prepare 2-3 of 245 mm LB plus ampicillin/Carbenicillin agarose plates. Each plate should have 350-400 mL of agarose in LB with 1:1000 of ampicillin/Carbenicillin

[STEPS]
SECTION: Generate barcode amplicon
1. qPCR reaction: 25ul/rxn

 
 
SECTION: Generate barcode amplicon
2. Purify with Zymo Clean & Concentrate with pooling 4 reactions together for one column: elute the column with 14 µL H2O. This is counting the volume needed for qubit and nanodrop. 
SECTION: Generate barcode amplicon
1.1. Thermocycling in a qPCR machine:
 
 
 
SECTION: Generate barcode amplicon
3. Quantify concentration of the final elution with Qubit and Nanodrop.

 

SECTION: Generating barcoded vectors using golden gate assembly
4. Setting up golden gate reactions:
 

 
 
 
SECTION: Generating barcoded vectors using golden gate assembly
4.1. Aliquot 25 µL per reaction to 8-well strip tube. 

SECTION: Generating barcoded vectors using golden gate assembly
4.2. Set the thermal cycler for the program below:
 
 
 
SECTION: Generating barcoded vectors using golden gate assembly
5. Make digestion mix:
 
 
 
SECTION: Generating barcoded vectors using golden gate assembly
5.1. Aliquot 5 µL to each golden gate reaction. 

SECTION: Generating barcoded vectors using golden gate assembly
5.2. Set the thermal cycler for the program below:
 
 
 
SECTION: Generating barcoded vectors using golden gate assembly
6. Purify with Zymo Clean & Concentrator -5 kit: pooling 3-4 reactions for each column with final elute to 10 µL. 

SECTION: Generating barcoded vectors using golden gate assembly
7. Measure the final elute with Nanodrop to look for normal purify curve, and quantify with Qubit: final reaction elution should be > 5ng/ul. The negative control should be < 5ng/ul
SECTION: Transformation with electro-competent cells and plating
8. 
 

SECTION: Transformation with electro-competent cells and plating
9. Pre-chill cuvettes on ice for at least 15 min. 

SECTION: Transformation with electro-competent cells and plating
10. Aliquot 875 µL of SOC media for each electroporation reaction to glass tubes. Pre-warm the tubes with media and an additional bottle of SOC in 37 °C.

SECTION: Transformation with electro-competent cells and plating
11. Prepare cells:

Thaw vials NEB electrocompetent cells (100ul/tube) on ice -immediately before the transformation. The cells start to die after ∼5 min.
SECTION: Transformation with electro-competent cells and plating
12. Add 25 µL thawed cells to each cuvette by tilting each cuvette towards you for easier pipetting. Stop at the 1st pipette stop to avoid injecting air bubbles to the cuvette. Air will cause your samples to “arc”.

SECTION: Transformation with electro-competent cells and plating
13. Add DNA to each sample to its cuvette into the cells with the cuvette tilted. Again, stop at the 1st pipette stop to avoid injecting air bubbles to the cuvette. Air will cause your samples to “arc”.

SECTION: Transformation with electro-competent cells and plating
14. Flick and tap cuvette after DNA is added with fingers, then lightly tap cuvettes a couple of times on the bench to mix. Put it back on ice before proceeding to the next samples.

SECTION: Transformation with electro-competent cells and plating
15. Chill all sample on ice for 30 min.

SECTION: Transformation with electro-competent cells and plating
16. Preparation for recovery:

While cells are chilling on ice.

Set up the machine: select for Manual at 2.0 ms- this will automatically set for 6 kV.
SECTION: Transformation with electro-competent cells and plating
17. Electroporation:

Wipe cuvette dry with kimwipe to remove condensation and ice.

SECTION: Transformation with electro-competent cells and plating
18. Start with the "H2O control". Electroporate each sample; make sure it makes connection to the metal. If a sample "arc", it is no longer good.

SECTION: Transformation with electro-competent cells and plating
19. Put each on ice before recovery them.

SECTION: Transformation with electro-competent cells and plating
20. Sample recovery:

Take the tubs with SOC from 37 °C.

SECTION: Transformation with electro-competent cells and plating
21. Transfer each of the cell + DNA (∼26-28u) to the labeled tubes. Put each cuvettes back on ice.

SECTION: Transformation with electro-competent cells and plating
22. Wash each curettes with 100 µL of warm SOC and transfer to recovery tubes.

SECTION: Transformation with electro-competent cells and plating
23. Put the recovery culture at 37 °C for 60 min with shaking.

SECTION: Transformation with electro-competent cells and plating
24. Preparing Dilution for plating:

Prepare 4 tubes (D0- D3) for each sample for a serial dilution with LB as below. Mix well.

 

 
 
SECTION: Transformation with electro-competent cells and plating
25. For each sample and Puc 19, label three 10cm LB + amp plates (D1-D3) and plate 100 µL from the D1-D3 corresponding tubes.

For negative control and H2O, plate 100 µL from the 1 mLrecovery culture. 

SECTION: Transformation with electro-competent cells and plating
26. Spread the plated culture with beads.
SECTION: Transformation with electro-competent cells and plating
27. Place the plates in 37 °C for 1200 min/faster growth.

SECTION: Transformation with electro-competent cells and plating
28. Plating the large plate: 

Pipette 2 mL-3 mL of recovery culture to each large LB+Carb plate, use the roller to spread the cell cultures. Leave the lids ajar slightly to dry the surface.

 

SECTION: Transformation with electro-competent cells and plating
28.1. Put all plates in 30 °C  for 960 min-1200 min incubation .

SECTION: Transformation with electro-competent cells and plating
28.2. Chill one tube of 50ml LB/plate in 4 °C for harvesting the next day. 

SECTION: Transformation with electro-competent cells and plating
29. Harvesting: 
The next day, count the colonies on  D2 and D3 dilution plates to estimate total colonies on each large plate. 

 

SECTION: Transformation with electro-competent cells and plating
29.1. The large plates must be harvested and prepped with a preferred plasmid Maxi prep kit immediately.