### **Library Creation Process SOP**

### **Transformation and Plating**

> Version Number: 3.0
>
> Start Production Date: 12-MAR-01
>
> Author: Amy Geotina
>
> Reviewed by: Sanjay Israni
>
> SOP Change Document: x

# Summary

This protocol is for the high-throughput electro transformation of pUC18
vector transformed into DH10B electro-competent cells and plated onto LB
Amp 100 X-gal plates.

# Materials & Reagents

> **[Materials/Reagents/Equipment]{.underline} [Vendor]{.underline}
> [Stock Number]{.underline}**

##### [Disposables]{.underline}

> LB Amp 100 X-gal plates (254mm) Teknova L133-A100X
>
> Nunc 384 well plate, cell culture Applied Scientific

##### [Stock Solutions]{.underline}

> LB Broth with 7.5% Glycerol Teknova 0181-G7.5
>
> SOC Media Teknova 0166-10

##### [Reagents]{.underline}

> ElectroMax Competent cells Gibco-BRL 18290-015

##### [Equipment]{.underline}

> Cell-Porator Life Technologies

# Procedure

**Transformation**

1.  Place one Micro-Electroporation Chamber and one 1.5ml eppendorf tube
    on ice for each library to be transformed and label each with the
    library name.

2.  Put 500ml SOC into one 14ml round bottom falcon tube per library and
    label with the library name.

3.  Add the following in order in the 1.5 ml eppendorf tube on ice:

> 1ul Ligation Reaction
>
> 25µl of DH10B ElectroMax DH10B Competent Cells (thaw on ice) pipette
> once or twice, mix with pipette tip gently
>
> Notes: This mixture must be kept on ice at all times.
>
> Dispose of any unused competent cells that have been thawed in
> autoclave waste.

4.  Fill the temperature control compartment of the Chamber Safe with a
    mixture of ice and water to keep the samples at approximately 4C.
    Place the Chamber Rack in the Chamber Safe.

5.  Pipette 25 ul of the ligation and cells between the bosses in a
    Micro-Electroporation Chamber. Be sure no air bubbles are present as
    that may cause arcing.

6.  Place each Chamber in a slot in the Chamber Rack carefully. If not
    using all of the slots fill the empty ones with blank Chambers. Use
    a Chamber only once and discard in autoclave waste.

7.  Close lid of the Chamber and secure the latch.

8.  Plug in the pulse cable into the right side of the Chamber.

9.  Turn the knob to point to the slot containing the Chamber to be
    electroporated.

10. Set the resistance on the Voltage Booster to 4 kiloohms; set the
    Pulse Control unit to LOW ohms for DC volts, the capacitance to 330
    uF and the change rate to Fast. Double check all settings.

11. Charge the unit to by setting the CHARGE ARM to Charge. Press UP on
    the voltage control button until the voltage is 5 to 10 volts higher
    than 410. Switch the CHARGE ARM to Arm and once the voltage drops to
    410 push the trigger button for 1 second. NOTE: The DC voltage
    display on the Pulse Control unit should read less than 10 volts
    after a pulse. If not, discharge the capacitor using the Down
    Button.

12. Turn the knob on the Chamber to point at the next sample and repeat
    steps 8 and 9.

13. After all samples have been electroporated, unplug the pulse cable
    and release the latch to remove the samples.

14. Immediately transfer samples from the microchamber into the falcon
    tube containing 500 ml of SOC before repeating steps 6-14 with the
    next four samples.

15. Incubate at 37 C on the wheel for 1 hour. Put on ice after 1-hour
    incubation period.

**Plating**

1.  While the transformation mixture is incubating, determine the number
    of LB/AMP/IPTG/X-gal bioassay plates necessary for plating. This
    number depends on the number of 384 well destination plates needed
    and transformation efficiency. Allow the plates to dry before use,
    place them slightly ajar in a 37^o^C incubator for up to 1.5 hours.

2.  After 1 hour, plate \~20ul of transformation mixture onto the
    desired number of bioassay agar plates. Use glass/metal spreaders to
    spread the reaction on the plates. Dip into ethanol and flame
    between plates to sterilize.

3.  Incubate the plates at 37^o^C for 17-20 hours on until average
    colony size is large enough for the colony picker to image.

**Fixing**

1.  Check deli for 384-well destination plates each day for plates that
    have been picked. Check for any plates that have picking IDs that
    belong to you. Hold each plate up to the light and mark on the
    underside of each plate any wells that did not grow (i.e. the well
    is clear). If more than five wells/quadrant are missing, the plate
    must be fixed. Do not seal plates with more than 20 wells where
    there was no growth. A perfect plate is preferred. If there are too
    many missing wells, have a new plate picked and use that one as a
    fix source.

2.  Remove liquid via vacuum system for each no growth well.

3.  Transfer the entire contents of a well from a fixing plate of the
    same library into the empty well. Change pipette tips between each
    sample. Fixing plates are labeled as such and are plates that do not
    have enough colonies to fill each of the 384 wells. You may need to
    create a fix plate by hand-picking with a sterile toothpick colonies
    that were not picked by the picker and incubating overnight.

**Sealing**

1.  After checking each destination plate to assure that they have no
    more than 20 wells without growth, place aluminum seal over each
    plate.

2.  Once sealed, place 2 labels on each plate. One on lid in A1 corner
    and the second on the base of the plate that corresponds to A1-H1.

3.  Scan plates into database.

4.  Place sealed and labeled plates in --80C freezer. Place each plate
    in numerical order in freezer rack that corresponds to library name.

5.  Write library and plate number entered in freezer and database onto
    the whiteboard in room 139.