Dec 03, 2019
Loop assembly using Labcyte Echo 550
- Susana Sauret-Gueto1,
- Anthony West2
- 1Plant Sciences, University of Cambridge, OpenPlant;
- 2previously at Earlham Institute, Norwich
- OpenPlant Project
Protocol Citation: Susana Sauret-Gueto, Anthony West 2019. Loop assembly using Labcyte Echo 550. protocols.io https://dx.doi.org/10.17504/protocols.io.8zvhx66
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 05, 2019
Last Modified: December 03, 2019
Protocol Integer ID: 29461
Keywords: typeIIS automation Loop
Abstract
Loop L1 and L2 type IIS assembly protocol, automated and minituarised for assembly of 500nl final volume reactions using the Labcyte Echo 550.
Materials
MATERIALS
BsaI - 5,000 unitsNew England BiolabsCatalog #R0535L
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
Sterile water
dATP, 100mM, 25uMolesPromegaCatalog #U1205
BSA, molecular biology grade, 20 mg/ml New England BiolabsCatalog # B9000S
10X NEB T4 DNA ligase bufferNew England Biolabs
384-Well Low Dead Volume (LDV) MicroplateCatalog #LP-0200
Tango BufferThermo Fisher ScientificCatalog #BY5
T4 DNA Ligase (5 U/µL)Thermo FisherCatalog #15224041
LguI (SapI) (5 U/µL)Thermo FisherCatalog #ER1932
384 PP plate labcyte
384 well skirted PCR plate (4ti-0384)
PCR foil seal (4ti-0550)
breath-easy sealing membrane (Z30059-1PAK)
Before using the Echo to do Loop assemblies, you need a list of all Loop reactions that needs doing, and all plasmids needed. Thus, make a csv files with all Loops that need doing. Separate Loop1 from Loop2. Examples:
| L1 construct | Accpetor plasmid (AP) | L0 plasmid in position 1 | L0 plasmid in position 2 | L0 plasmid in position 3 | L0 plasmid in position 4 | L0 plasmid in position 5 | |
| L1_001-Ck1 | pCk1 | PROM5_a | CDS_a | 3TERM_a | |||
| L1_002-Ck2 | pCk2 | PROM5_a | CDS12_b | CTAG_b | 3TERM_a | ||
| L1_003-Ck2 | pCk2 | PROM5_b | CDS12_b | CTAG_b | 3TERM_a | ||
| L1_004-Ck2 | pCk2 | PROM_c | 5UTR_c | CDS12_b | CTAG_b | 3TERM_a |
Example for L1 Loop
| L2 construct | Accpetor plasmid (AP) | L1 plasmid in position 1 | L1 plasmid in position 2 | L1 plasmid in position 3 | L1 plasmid in position 4 | |
| L2_001-CsA | pCsA | L1_001-Ck1 | L1_002-Ck2 | L1_005-Ck3 | L1_006-Ck4 | |
| L2_002-CsA | pCsA | L1_001-Ck1 | L1_003-Ck2 | L1_005-Ck3 | L1_006-Ck4 |
Example for L2 Loop
Next create LDV source plate that contain all plasmids needed but the APs.
For L1 Loops, have a LDV source plate with the libary of L0 parts.
For L2 Loops, have a LDV source plate with the library of L1 plasmids.
Use Echo calibration 384LDV_AQ_B2 for these plates.
LDV plates working range is~ 3-12 ul (maximum volume is 12 ul and dead volume is 3 ul).
Thus, fill in the wells wtih 10 ul of a given plasmid.
Source plasmids final concentration is 15 nM.
For a final concentration of 15 nM, the concentration in [ng/ul] equals N (the length in bp of the plasmid) divided by 110. This is an approximation of the formula:
15∙10^(-9)mol/L x ((607.4 x N ) + 157.9)g/mol x 10^(-6)L/μL x 10^9ng/g = concentration (ng/μL)
This plate can be sealed and kept at -20C, for reuse in the future as many times as necessary, and empty wells can be filled in with new plasmids.
Keep a csv files with the LDV plate source well and sample name and update as you fill in new positions.
Next create PP source plate with the APs and MM and water.
For L1 Loops, have a PP source plate with the pCk APs and L1-MM-BsaI.
For L2 Loops, have a PP source plate with the pCs APs and L2-MM-SapI.
Use Echo calibration 384PP_AQ_BP2 for these plates.
PP plates working range is ~ 15-65 ul (maximum volume is 65 ul and dead volume is 15 ul).
Thus, fill in the wells wtih ~30-50 ul of a given plasmid.
MM contains enzymes, BSA and glycerol, and has a higher tendency to creep up the walls of the well than water, thus the dead volume is higher, at around 18 ul (always check the dead volume for your MM). The dead volume is the volume at which the transducer of the Echo can not detect the top of the meniscus anymore and thus can not focus to transfer droplets by accoustic energy.
Prepare a source 384 PP plate with APs (pCks, pCs) at a final concentration of 7.5 nM and Loop plasmids with spacers (pCk1-spacer, pCk2-spacer,....) at a final concentration of 15 nM.
Use two wells in this plate for water and for the master mix (MM).
This plate can sealed, kept at -20C, and reused, and every time a new Loop-Echo assembly needs doing, choose a new pair of wells for water and MM. Alternatively have two different PP plates, one for the APs, and one for the water and MM.
Keep a csv files with the PP plate source well and sample name and update as you fill in new positions.
Final volume of L2-Loop reactions at the Echo is 500 nl, with 250 ul of MM (see next step on preparing MM) and 250 ul of plasmids (50 ul of each one of the 5 plasmids), as follows:
| Plasmid | Volume | |
| pCk2 | 50 nl | |
| L1_001-Ck1 | 50 nl | |
| L1_002-Ck2 | 50 nl | |
| L1_004-Ck3 | 50 nl | |
| L1_005-Ck3 | 50 nl | |
| L2_MM-SapI | 250 nl | |
| Final volume | 500 nl |
Example of volumes dispensed by Echo for to assemble L2_001-CsA
For L2 Loops, prepare MM as follows:
| Volume for 1 reaction in 1 well in a 384 PCR plate | Volume for 96 reactions in a 384 PCR plate | ||
| Water | 100 nl | 9.6 ul | |
| 10x Tango buffer (Thermo Fisher) | 50 nl | 4.8 ul | |
| 1 mg/mL bovine serum albumin (NEB) | 25 nl | 2.4 ul | |
| 10mM ATP (SIGMA) | 50 nl | 4.8 ul | |
| T4 DNA ligase (5 U/µL) (Thermo Fisher) | 12.5 nl | 1.2 ul | |
| SapI (LguI) (5 U/µL) (Thermo Fisher) | 12.5 nl | 1.2 ul | |
| Final volume | 250 nl | 24 ul |
L2-MM-SapI
To prepare the MM always consider the volume you need for the plate reactions plus the dead volume of the MM solution in a PP plate.
Always prepare the MM in an eppendorf tube first, mix by pipetting and then transfer the MM to the PP-well. This is to ensure all components of the MM are well mixed.
Final volume of L1-Loop reactions at the Echo is 500 nl. In this case, because number of plasmids needed is variable (4-7) the MM volume needs to change depending on that. Thus, if we want to do different L1-Loops with different number of plasmids, we can assume a 6 plasmids for all, and use water for the positions without a plasmid as whown below.
| L1-Loop with 6 plasmids | L1-Loop with 5 plasmids | L1-Loop with 4 plasmids | ||||
| pCk2 | 50 nl | pCk2 | 50 nl | pCk2 | 50 nl | |
| PROM_c | 50 nl | PROM5_b | 50 nl | PROM5_a | 50 nl | |
| 5UTR_c | 50 nl | CDS12_b | 50 nl | CDS_a | 50 nl | |
| CDS12_b | 50 nl | CTAG_b | 50 nl | 3TERM_a | 50 nl | |
| CTAG_b | 50 nl | 3TERM_a | 50 nl | water | 100 nl | |
| 3TERM_a | 50 nl | water | 50 nl | |||
| L1_MM6-BsaI | 200 nl | L1_MM6-BsaI | 200 nl | L1_MM6-BsaI | 200 nl | |
| Final volume | 500 nl | Final volume | 500 nl | Final volume | 500 nl | |
Example of volumes dispensed by Echo in different L1-Loop reactions
For L1 Loops, prepare MM for maximum of 6 parts as follows:
| Volume for 1 reaction in 1 well in a 384 PCR plate | Volume for 96 reactions in a 384 PCR plate | ||
| Water | 100 nl | 9.6 ul | |
| 10x T4 ligase buffer (NEB) | 50 nl | 4.8 ul | |
| 1 mg/mL bovine serum albumin (NEB) | 25 nl | 2.4 ul | |
| T4 DNA ligase at 400 U/μL (NEB) | 12.5 nl | 1.2 ul | |
| 10 U/μL BsaI (NEB) | 12.5 nl | 1.2 ul | |
| Final volume | 200 nl | 19.2 ul |
L1-MM6-BsaI
To prepare the MM always consider the volume you need for the plate reactions plus the dead volume of the MM solution in a PP plate.
Always prepare the MM in an eppendorf tube first, mix by pipetting and then transfer the MM to the PP-well. This is to ensure all components of the MM are well mixed.
Generate a csv file to upload in the Labcyte cherry pick software with the following information:
| Source Plate Name | Source Well | Destination Well | Sample Name | Transfer Volume | Source Plate Type | Destination Plate Name | Destination Plate Type |
Generation of the csv files can be done as more convinient, with an excel template or a phyton script, or ...
Previous to running and Echo experiment, make sure the information in the csv file is correct and the Echo is running what you intend to run. You can simulate the running at the Echo.
Loop assembly using the Echo:
Load csv file in the cherry pick.
Get LDV and PP source plates:
LDV plate: defrost, spin down and keept at RT.
PP plate: defrost, spin down, keep at RT. Prepare MM, spin and keep at RT.
Get the destination plates: skirted 384-PCR plates
Run the L1 or L2 Loop assemblies at the Echo.
If there are any exceptons, proceed to re-run those dispensations.
Seal the 384-PCR plate with a PCR foil seal and spin it down.
Place the 384-PCR plates at the termocycler and run the follwoing cycling conditions:
Assembly: 26 cycles of 37 °C for 00:03:00 and16 °C for 00:04:00 .
Termination and enzyme denaturation: 50 °C for 00:05:00 and80 °C for 00:10:00 .
Transform the Loop assemblies with highly competent cells (10^8) as follows:
Note: we use homemade Top10 HS higly competent cells.But any other ones higly competent will work as well.
Add 2 ul of cells to each well
Incubate 00:15:00 on ice
Heat shock at 42 °C for 00:00:40 in a thermocycler
Incubate 00:02:00 on ice
Add 15 u of LB to each well
Seal the plate with a breath-easy sealing membrane
Incubate the plate at 37 °C on a shaker for 01:00:00
Plate(*) the ~ 18ul fo the reaction in LB agar plates with with either:
for L1 kanamycin 50 μg/mL and X-gal 40 ng/ mL
for L2 spectinomycin 100 μg/mL and X-gal 40 ng/ mL
Incubate plates 00:15:00 O/N at 37 °C
All white colonies have an insert inside the AP vector. The efficiency of the assemnmbly is really high, and we normally just check 2 colonies.
(*) Alternatively, one can use the Echo to tranfer the transformed cells onto a plate. It needs transfering of the transformation to a PP plate and spotting with the Echo onto an omnitray.