**** New Dual positive (NPTII & Sl. pFAST) PLUS anti-selection (FCY-UPP) iCas9 (pICSL22022)/Cas12a (pICSL22023) acceptor constructs added. Both constructs contain an AmilRFP negative selection cassette and accept guides in all positions (1-7) via Bpi1. ****
If you are having problems with your Golden Gate (Type II) assembly method, the following hints and tips should help solve most of the ones I most commonly encounter. Before making an appointment to discuss any Golden-Gate (Type II) assembly issues with me in person, please ensure that you have tried and tested all of the below mentioned reaction parameters. If however you have and things are still not working as expected, please just drop me an email and make an appointment to discuss things in further detail.
ONLY use the NEB ligase Buffer and the NEB T4 DNA ligase (low concentration #M0202S/L).
ONLY use Thermofisher restriction enzymes (Bpi1 #ER1011 and Eco31I #ER0292).
ALWAYS gel extract any PCR products. I HIGHLY recommend using Zymoclean DNA recovery Kit (Cambridge Bioscience #D4008).
MIDI-Prep ALL plasmids used for L1 and L2 assemblies. Although Mini-prep DNA quality is good enough for sequencing, it is NOT generally good enough for complex DNA assemblies.
ALWAYS use 15ul reaction volumes (unless specifically instructed e.g. sequential Cas9 guide insertion protocol).
ALWAYS use 0.5ul (5U restriction/200U Ligase) of both restriction and ligation enzymes.
ALWAYS individually calculate the relative (ng) proportions needed for each reaction part/module. Each part/module needs to be at a 2:1 ratio, relative to the acceptor module being used. Assuming you are using the recommended (100ng) of an acceptor vector, a simple calculation to determine the amounts of each part/module needed is: module size (bp)/acceptor size (bp) x200 = ng of part needed.
ALWAYS use the long (3mins 37°C, 4mins 16°C x26) cycling protocol.
When designing custom overhangs for module/part domestication, carefully look at the overhangs already being used in the cloning strategy and ensure that any new overhangs ALWAYS have unique bases in 3 out of the 4 positions (relative to any other individual overhangs being used).
It can be problematic if a required sequence has 6 (or more) BsaI/BpiI sites that need to be removed for domestication. If this is the case then it may first be neccesary to chop the sequence into smaller parts (i.e. 3x2) and then later assemble the required sequence from the smaller number of fragments. In order to achieve this new, custom (L0) acceptor vectors will usually first need to be made. We do however already have custom L1 (CDs) acceptors (AATG-GCTT and AATG-TTCG) for the assembly of multiple, custom L0 parts.
Transformation plates dominated by BLUE colonies generally implicate problems relating to the restriction enzymes being used. NO colonies on the transformation plates, generally implicates a problem relating to the ligation enzyme being used.