— Jacob Anderson 2018/11/21 12:05

Guidelines and instructions are provided to perform a MultiSite Gateway® Pro LR recombination reaction between suitable supercoiled entry clones and a supercoiled destination vector using LR Clonase™ II Plus enzyme mix. We recommend including and negative control (no LR Clonase™ II Plus) and one or more positive control reactions.

You must use LR Clonase™ II Plus enzyme mix to catalyze the MultiSite Gateway® Pro LR recombination reaction.

Depending on your downstream applications, you will need to provide an appropriate Gateway® destination vector for the LR recombination reaction. You may use any destination vector of choice that contains attR1 and attR2 sites.

You cannot use pDEST R4-R3 from the MultiSite Gateway® Three-Fragment Vector Construction Kit, because the attR3 and attR4 sites are incompatible with recombination with attL1- and attL2-containing entry clones.

You will need to have purified midiprep plasmid DNA of each entry clone to perform the MultiSite Gateway® LR recombination reaction.

You will need the following materials before beginning

• LR Clonase 2 plus enzyme mix

• 2 µg/mL Proteinase K Solution

• OneShot Mach1 T1 Chemically competent E. coli

• S.O.C. Medium * Unordered List Item

• Midiprep-purified plasmid DNA of your entry clones

• * Important – you will need to add plasmid DNA from two, three, or four entry clones to the MultiSite Gateway® Pro LR reaction. Make sure that the plasmid DNA for each entry clone is sufficiently concentrated such that the total amount of entry clone plasmid DNA added to a 8µL MultiSite Gateway® Pro LR reaction does not exceed 7 µL total.

• Purified plasmid DNA of you destination vector

• 1X TEA buffer, pH 8.0

• Bacterial growth media for expression

• Selective LB agar plates containing 50-100 µg/mL antibiotic, depending on the resistance gene present in your destination vector

1. Add the following components to 1.5 mL microcentrifuge tubes at room temperature and mix.

• Entry clone (10 fmoles each) - 1-7 µL

• Destination vector (20 fmoles) - 1 µl

• 1X TEA Buffer, pH 8.0 - to 8 µL

2. Remove the LR Clonase™ 2 plus enzyme mix from the -20°C and thaw on ice (~2min)

3. To each sample above, add 2µL of LR Clonase™ 2 Plus enzyme mix. Mix well by vortexing briefly twice. (2 seconds each time)

4. Return LR Clonase™ 2 Plus enzyme mix to -20°C immediately after use.

5. Incubate reactions at 25°C for 16 hours

6. Add 1µL of the proteinase K solution to each reaction. Incubate for 10 minutes at 37°C.

NEXT STEPS If your recombination reaction was successful (i.e. provided the expected number of colonies) you may express your clone in the system appropriate for your destination vector. Depending on the length of the inserts in your clone and the presence of specific primer binding sites on your destination vector, you may sequence your expression clone.

If your recombination reaction was not satisfactory (i.e. resulted in fewer than expected or no colonies) you should perform the control reactions to troubleshoot your MultiSite Gateway® Pro LR recombination reaction.

Use the following formula to convert fentomoles (fmoles) of DNA to nanograms (ng) of DNA:

ng = (x fmoles)(N)(660fg/fmoles)(1ng/10^6 fg) = ### ng, where x is the number of fmoles and N is the size of the DNA in bp. For an example, see below.

In this example, you need to use 50 fmoles of an attB PCR product in the BP reaction. The attB PCR product is 2.5 kb in size. Calculate the amount of attB PCR product required for the reaction (in ng) by using the equation above:

(50 fmoles)(2500bp)(660fg/fmoles)(1ng/10^6 fg) = 82.9 ng

list the full manufacturer name and manufacturer part number for all reagents, and then for all equipment. If these already have wiki pages you should be able to get the numbers there. PART NUMBERS will always have two sections, first Reagents and then Equipment. Please place these items in 2 tables.