Manual LIC Reaction Setup

Overview of Manual LIC Reaction Setup

Preparation:

• Turn heat block in Robot Room to high setting. This should go to 75ºC. 
• If you are processing more than two plates they will have to be spaced at least 20 minutes apart. The heat block only holds two plates at a time and this is the rate limiting step. 
• Place the thermal chiller/exchanger in slot A3 on the Biomek. Pre-chill the quarter vertical reservoir for LIC Rxn by placing it on ice. 
• Make LIC Reaction mix as follows for one plate: 
• 465 µl         10x LIC Ac Reaction Buffer 
• 465 µl         25mM dCTP (dCTP comes 100mM, so every 1 µl is diluted with 3 µl of water) 
• 228 µl         100mM DTT solution 
•   61 µl         Water 
• 250 units     T4 DNA Polymerase (Novagen-LIC quality) 
  
• Keep this mixture on ice and add the T4 just before use. 
• Label an MJ Thermocycler plate "(Plate ID) LIC (Today's Date)".

Procedure:

• smLIC, carried out on the Biomek, arrays 10.4 ?l of LIC reaction mix into the labeled MJ Thermocycler plates. This method will array two plates at a time. While this method is running, begin running the method in step 2.
• PCRf1-1, carried out on the Multimek, prepares a portion of the "(Plate ID) PCR Frags (Today's Date)" by diluting it with water and placing that dilution in a temporary plate. 
• LICRx5, carried out on the Multimek, will combine the dilution from step 2 with the LIC reaction mix from step 1. 
• LICRx2, carried out on the Multimek, will overlay mineral oil on the labeled LIC plates.
• The plates are left at room temperature for 30 minutes, then place on the heat block at 75ºC for 20 minutes. 
• Following the heating process the LIC plates are stored in the refrigerator at 4ºC until needed.
  
  
• Next Step: Manual Annealing Reaction Setup

LIC Reaction Setup (LICRx HT.smt)

Method designed for processing multiples of two plates

Before automation run -

• NOTE: limiting step at HeatBlock. Plates must be interleaved at 20 minute intervals.
• Turn heat block on high setting (75°C). 
• Place thermal chiller/exchanger in slot A3 on Biomek. 
• Turn on refrigeration unit or use ice for cooling elements on Biomek. 
• Prepare LIC reaction mix immediately before running method smLIC. 
  • Remind user that ice will be required for this method. Keep reaction mix on ice until use.
    To a microcentrifuge tube add (enough for one 96-well plate)--- 
                465µl               10x LIC Ac Reaction Buffer 
                465µl               25mM dCTP 
                228µl               100mM DTT Solution 
                61µl                 H₂O 
                250 units          T4 DNA Polymerase (Novagen-LIC quality) 
    Place LIC reaction mix into 'Quarter Vertical' reservoir--
    Place 'Quarter Vertical' reservoir onto reservoir holder-- 
    Place reservoir holder onto thermal chiller/exchanger in slot A3.

Method Sequence

• PCRf1-1-method prepares a 1:1 dilution of the PCR fragment in distilled water. Diluted fragment mix is prepared in a temporary storage plate. Ratio may be adjusted to compensate for PCR fragment concentration. 
• smLIC-arrays 10.4 µl of LIC reaction mix into the LIC reaction plate. 
• LICRx5-method transfers 28 µl of diluted PCR fragment to the LIC reaction mix plate and pipets up and down several times to mix. 
• LICRx2-method overlays 16 µl of mineral oil 
• Let LIC plate stand @ room temperature for 30 minutes 
• Transfer LIC plate to heatblock for 20 minutes @ 72ºC or 75ºC

Multimek method PCRf1-1

Biomek method SmLIC

Multimek method LICRx5

Multi mekmethod LICRx2