iGEM 2016 — Team Lethbridge

[![](https://static.igem.org/mediawiki/2016/8/8b/HQ_page_logo.jpg)](https://2016.igem.org)

# Team:Lethbridge/Notebook

Lethbridge iGEM 2016

[Lethbridge iGEM](https://2016.igem.org/Team%3ALethbridge)

* [Project](#!)

+ [Description](https://2016.igem.org/Team%3ALethbridge/Description)
+ [Design](https://2016.igem.org/Team%3ALethbridge/Design)
+ [Experiment](https://2016.igem.org/Team%3ALethbridge/Experiments)
+ [Database](https://2016.igem.org/Team%3ALethbridge/Database)
+ [Notebook](https://2016.igem.org/Team%3ALethbridge/Notebook)
+ [Parts](https://2016.igem.org/Team%3ALethbridge/Parts)
+ [Safety](https://2016.igem.org/Team%3ALethbridge/Safety)
+ [RNAi](https://2016.igem.org/Team%3ALethbridge/RNAi)

* [Practices](#!)

+ [Integrated Practices](https://2016.igem.org/Team%3ALethbridge/Integrated_Practices)
+ [Contact](https://2016.igem.org/Team%3ALethbridge/HP/Silver#intro)
+ [Interviews](https://2016.igem.org/Team%3ALethbridge/HP/Silver#interviews)
+ [Literature Review](https://2016.igem.org/Team%3ALethbridge/HP/Silver#litrev)
+ [Ridealongs](https://2016.igem.org/Team%3ALethbridge/HP/Gold#ridealong)
+ [Protocol](https://2016.igem.org/Team%3ALethbridge/HP/Gold#protocol)
+ [Sampling](https://2016.igem.org/Team%3ALethbridge/HP/Gold#sampling)

* [Results](https://2016.igem.org/Team%3ALethbridge/Results)
* [Team](#!)

+ [Members](https://2016.igem.org/Team%3ALethbridge/Team)
+ [Collab](https://2016.igem.org/Team%3ALethbridge/Collaborations)
+ [Attribution](https://2016.igem.org/Team%3ALethbridge/Attributions)
+ [Sponsors](https://2016.igem.org/Team%3ALethbridge/Sponsors)

* [Achievements](https://2016.igem.org/Team%3ALethbridge/Achievements)

*menu*

* + Project

    - [Description](https://2016.igem.org/Team%3ALethbridge/Description)
    - [Design](https://2016.igem.org/Team%3ALethbridge/Design)
    - [Experiments](https://2016.igem.org/Team%3ALethbridge/Experiments)
    - [Database](https://2016.igem.org/Team%3ALethbridge/Database)
    - [Notebook](https://2016.igem.org/Team%3ALethbridge/Notebook)
    - [Parts](https://2016.igem.org/Team%3ALethbridge/Parts)
    - [Safety](https://2016.igem.org/Team%3ALethbridge/Safety)
    - [RNAi](https://2016.igem.org/Team%3ALethbridge/RNAi)
* + Practices

    - [Integrated Practices](https://2016.igem.org/Team%3ALethbridge/Integrated_Practices)
    - [Intro](https://2016.igem.org/Team%3ALethbridge/HP/Silver#intro)
    - [Interviews](https://2016.igem.org/Team%3ALethbridge/HP/Silver#interviews)
    - [Literature Review](https://2016.igem.org/Team%3ALethbridge/HP/Silver#litrev)
    - [Ridealongs](https://2016.igem.org/Team%3ALethbridge/HP/Gold#ridealong)
    - [Protocol](https://2016.igem.org/Team%3ALethbridge/HP/Gold#protocol)
    - [Sampling](https://2016.igem.org/Team%3ALethbridge/HP/Gold#sampling)
* [Results](https://2016.igem.org/Team%3ALethbridge/Results)
* + Team

    - [Members](https://2016.igem.org/Team%3ALethbridge/Team)
    - [Collaborations](https://2016.igem.org/Team%3ALethbridge/Collaborations)
    - [Attribution](https://2016.igem.org/Team%3ALethbridge/Attributions)
    - [Sponsors](https://2016.igem.org/Team%3ALethbridge/Sponsors)
* [Achievements](https://2016.igem.org/Team%3ALethbridge/Achievements)

## Notebook

##### The various facets of our lab work were divided between several key areas. Please explore!

[Nanopore Microbiome Analysis](https://2016.igem.org/Team%3ALethbridge/Notebook#microbiome)

[Single-domain antibody development system](https://2016.igem.org/Team%3ALethbridge/Notebook#pathogen)

#### Nanopore Microbiome Analysis

| Date | Experiment |
| --- | --- |
| [2016/08/16](#Entry1) | DNA extraction protocol |
| [2016/08/16](#Entry2) | Multiple displacement amplification of EMS - Week #1 |
| [2016/08/24](#Entry3) | 16S rRNA gene amplification from EMS samples (week #4) |
| [2016/08/31](#Entry4) | Optimization of PCR amplification using various rDNA gene primer pairs |
| [2016/08/31](#Entry5) | Large scale PCR amplification using 16S and 18S rRNA |
| [2016/10/4](#Entry6) | DNA extraction - Week #4 samples |
| [2016/10/5](#Entry7) | Amplify 1N4, 2N4, 3N4, Pseud., virus control, E. coli control |

DNA extraction protocol

###### 2016/08/16

Protocol for the extraction of DNA from samples taken from EMS ride-alongs.

1. Ethanol evaporation for approximately 2 hours on the rotary evaporator on high heat, under vacuum and high speed.
2. Add 400 µl of alkaline lysis buffer (200 mM KOH, 50 mM DTT).
3. Mix by inverting tube several times.
4. Incubate on heat block at 65 °C for 10 minutes.
5. Centrifuge at 17,000 x g for 1 minute.
6. Pipette liquid volume (as much as possible) into new 1.5 mL microcentrifuge tube (~300 µl)
7. Add 1/10 of the volume (~30 µl) of 3 M sodium acetate (pH 5.3) to each tube.
8. Add 5 µl of 0.5% LPA (linear polyacrylamide) carrier to each tube.
9. Add 750 µl of cold 100% ethanol to each tube, mix by inverting and place tubes on ice for approximate 10 minutes.
10. Centrifuge at 17,000 x g for 5 minutes.
11. Remove supernatant.
12. Wash with 500 µl of 70% ethanol and mix gently. Push centrifuge and remove supernatant.
13. Repeat wash step (step 12)
14. Vacuum evaporate the ethanol under high heat for 10 minutes.
15. Resuspend in 20 µl of filtered MilliQ water.

#### Single Domain Antibody Development System

| Date | Experiment |
| --- | --- |
| [2016/05/01](#Entry8) | Transformation of plasmids into NEB5α cells |
| [2016/05/09](#Entry9) | Set up of O/N cultures to isolate plasmids for SLIC |
| [2016/05/13](#Entry10) | PCR amplification of RNAPα::taget scaffold gBlock |
| [2016/05/18](#Entry11) | PCR amplification of pSB1T3 and RNAPα::target for T4 DNA polymerase chewback reaction |
| [2016/05/19](#Entry12) | SLIC assembly of pSB1T3-RNAPα::target |
| [2016/05/26](#Entry13) | PCR amplification for SLIC assembly of pSB3C5\_Cl::Nb\_Anti-GFP Nb |
| [2016/06/1](#Entry14) | SLIC assembly of pSB3C5\_Cl::Nb\_Anti-GFP Nb |
| [2016/06/01](#Entry15) | Amplification of GFP (BBa\_E0040) using oNb52 and oNb47 |
| [2016/06/02](#Entry16) | Colony PCR of potential pSB3C5\_Cl::Nb\_Anti-GFP Nb clones |
| [2016/06/05](#Entry17) | Screening of pSB1T3\_RNAPα::target clones using BglII restriction digest |
| [2016/06/25](#Entry18) | Single-domain antibody library cloning |
| [2016/07/25](#Entry19) | Restriction digest of single-domain antibody (sdAb) scaffold |
| [2016/08/3](#Entry20) | Generation of CDR1-2 library by oligo assembly and PCR |
| [2016/08/4](#Entry21) | Generation of CDR3 libraries |
| [2016/08/08](#Entry22) | Ligation of CDR1-2 and sdAb scaffold |
| [2016/08/10](#Entry23) | CDR3 NgoMIV digestion |
| [2016/08/11](#Entry24) | KpnI and NgoMIV digests of sdAb scaffold and CDR1-2 |
||  |  |
| --- | --- |
| [2016/08/16](#Entry25) | Ligation PCR of CDR3s and sdAb/CDR1-2 |
| [2016/08/18](#Entry26) | CloneJet cloning of sdAb scaffold/CDR1-2\_CDR3 ligation PCRs |
| [2016/08/31](#Entry27) | PCR amplification and overlap extension PCR assembly of sdAb library parts |
| [2016/09/07](#Entry28) | Overlap extension PCR assembly of sdAb library |
| [2016/09/07](#Entry29) | Overlap extension PCR of sdAb 5' CDR1-2 with CDR3 sdAb 3' |
| [2016/09/26](#Entry30) | Scale up PCR amplification of sdAb libraries |
| [2016/09/27](#Entry32) | PCR amplification of pSB3C5\_Cl::Nb backbone for sdAb library SLIC assembly |
| [2016/09/28](#Entry33) | SLIC assembly of sdAb library into pSB3C5\_Cl::Nb |
| [2016/09/29](#Entry34) | sdAb library SLIC assembly electroporation results |
| [2016/10/07](#Entry35) | Colony PCR screening of pSB4K5\_pBAD RNAPα |
| [2016/10/12](#Entry36) | PCR amplification of pSB3K5 for pBAD-RNAPα and B2HR ver 2-1 cloning |
 [2016/10/13](#Entry37) | PCR-amplification optimization of B2HR ver 2-1 gBlock from pJET clone |
 [2016/10/17](#Entry38) | PCR-amplification of BioBrick parts for cloning into pSB1C3 and pSB3K5 | [2016/10/18](#Entry39) | Restriction digest of B2HR ver 2.1 and RNAPα pBAD for cloning into pSB1C3 and PSB3K3 ||

Transformation of plasmids into NEB5α cells

###### 2016/12/29

The plasmids obtained from the registry were transformed into NEB5α cells.

1. Resuspend DNA from kit plate in 10 µl of sterile MilliQ water

| Plasmid | Well | Kit plate (year) |
| --- | --- | --- |
| pSB3K3 | 6F | Kit plate 4 (2016) |
| pSB3C5 | 4D | Kit plate 4 (2015) |
| pSB1T3 | 8B | Kit plate 4 (2015) |
| pSB4K5 | 6H | Kit plate 4 (2016) |

2. Add 1 µl of DNA to high efficiency NEB5α cells
3. Incubate on ice for 30 minutes
4. Heatschok at 42 °C for 45 seconds. Keep reactions on ice for the following 5 minutes
5. Add 400 µl of LB media and incubate at 37 °C for 1 hour
6. Plate 200 µl of each reaction in the LB plates containing the respective antibiotic

Previous
Next

PCR amplification of pSB3C5\_Cl::Nb and Anti-GFP Nb for SLIC assembly

###### 2016/05/11

To linearize the backbone (pSB3C5\_Cl::Nb) use primers BioBrick suffix forward (5' TACTAGTAGCGGCCGCTGCAG 3') and oNb46 (5' CATACTCGAGGCTGCGGCGC 3'). The amplification of anti-GFP Nb (BH-GBP3 gBlock) was done using oNb51 (5' GCGCCGCAGCCTCGAGTATGGATGTGCAACTGCAAGAATCAG 3') and BioBrick suffix reverse (5' CTGCAGCGGCCGCTACTAGTA 3').

Table 1. Reagents and the volumes used to set up various PCR reactions

| Reagent | Volume (µl) |
| --- | --- |
| 2x Phusion MM, HF buffer | 25 |
| Forward primer, 10 µM | 2.5 |
| Reverse primer, 10 µM | 2.5 |
| DNA template (10 ng) | 1 |
| MilliQ water | 19 |

Table 2. PCR cycle used for the amplification of different DNA fragments

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 15 | 30 |
| Annealing | 58 | 20 | 30 |
| Extension | 72 | 45 | 30 |
| Final extension | 72 | 300 | 1 |

Previous
Next

<

PCR amplification of pSB1T3 and RNAPα::target for SLIC

###### 2016/05/13

The plasmid was linearized using the BioBrick prefix forward (5' GAATTCGCGGCCGCTTCTAGAG 3') and BioBrick reverse suffix, while the gBlock (RNAPα::target) was amplified using BioBrick prefix reverse (5' CTCTAGAAGCGGCCGCGAATTC 3') and BioBrick suffix forward.

Table 1. Reagents and the volumes used to set up various PCR reactions

| Reagent | Volume (µl) |
| --- | --- |
| 2x Phusion MM, HF buffer | 25 |
| Forward primer, 10 µM | 2.5 |
| Reverse primer, 10 µM | 2.5 |
| DNA template (10 ng) | 1 |
| MilliQ water | 19 |

Table 2. PCR cycle used for the amplification of different DNA fragments

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 15 | 30 |
| Annealing | 71.7 | 20 | 30 |
| Extension | 72 | 45 | 30 |
| Final extension | 72 | 300 | 1 |

Note: Template used for the amplification of RNAPα::target was the wrong one. Use gBlock of pJET clone in future experiments.

Previous
Next

Large scale PCR of pSB1T3 and RNAPα::target scaffold to generate more DNA for chewback reactions.

###### 2016/05/18

The previous PCR did not yield enough DNA to carry out the second step of SLIC, a chewback reaction. In this step, the DNA is made single stranded by taking advantage of the exonuclease activity of T4 DNA polymerase.

Table 1. Reagents and the volumes used to set up various PCR reactions

| Reagent | Volume (µl) |
| --- | --- |
| 5x HF Phusion Buffer | 10 |
| Phusion (2U/µl) | 1 |
| dNTPs, 10 mM | 1 |
| Reverse primer, 10 µM | 2.5 |
| Forward primer, 10 µM | 2.5 |
| DNA template (10 ng) | 1 || MilliQ water | 32 |

Table 2. PCR cycle used for the amplification of different DNA fragments

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 15 | 30 |
| Annealing | 71.7 | 20 | 30 |
| Extension | 72 | 45 | 30 |
| Final extension | 72 | 300 | 1 |

Note: Template used for the amplification of RNAPα::target was the wrong one. Use gBlock of pJET clone in future experiments.

Previous
Next

SLIC assembly of pSB1T3\_RNAPα::target.

###### 2016/05/19

Table 1. Quantification of the purified PCR products using a BioDrop

| Sample | [DNA](ng/µl) | A260/280 |
| --- | --- | --- |
| pSB1T3 SLIC PCR | 78.18 | 1.854 |
| RNAPα::target SLIC PCR | 50.24 | 1.844 |

Chewbackk reactions of 3' ends using T4 DNA polymerase

| Reagent | Volume (µl) |
| --- | --- |
| 10x NEBuffer 2.1 | 2 |
| DNA fragments (~ 1000 ng) | 16 |
| MilliQ water | 0 |
| T4 DNA polymerase (0.3 U/µl) | 1 |
| DpnI (10 U/µl)/td> 1 | |

Incubate reactions for 30 minutes in a 37 °C waterbath

Add 2 µl of 10 mM dCTP to arrest the reaction. Inmediately transfer the tubes to an ice box

Assembly of DNA fragments

| Fragment | DNA size (bp) | Relative size | Equivalent DNA amount (ng) | [Chewback] (ng/µl) | Amount of chewback needed (µl) |
| --- | --- | --- | --- | --- | --- |
| pSB1T3 SLIC PCR | 2497 | 1 | 1000 | 60 | 16 |
| RNAPα::target SLIC PCR | 1028 | 0.41 | 410 | 40 | 10.25 |

Mix the required DNA amounts in a microcentrifuge tube and incubate for 10 minutes at 37 °C in a waterbath.

Clean up reaction using the BioBasic enzymatic protocol.

Electroporate 100 ng of DNA into fresh electrocompetent E. coli BL21(DE3) gold.

Previous
Next

PCR amplification for SLIC assembly of pSB3C5\_Cl::Nb\_Anti-GFP Nb

###### 2016/05/26

More PCR product is needed to do the chewback reaction. Large scale PCRs were set up for this purpose

Table 1. Reagents and the volumes used to set up various PCR reactions

| Reagent | Volume (µl) |
| --- | --- |
| 2x Phusion MM, HF buffer | 75 |
| Reverse primer, 10 µM | 7.5 |
| Forward primer, 10 µM | 7.5 |
| DNA template (10 ng) | 3 || MilliQ water | 57 |

Table 2. PCR cycle used for the amplification of different DNA fragments

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 15 | 30 |
| Annealing | 58 | 20 | 30 |
| Extension | 72 | 45 | 30 |
| Final extension | 72 | 300 | 1 |

Previous
Next

SLIC assembly of pSB3C5\_Cl::Nb\_GBP3

###### 2016/06/01

Table 1. Quantification of the purified PCR products using a BioDrop

| Sample | [DNA](ng/µl) | A260/280 |
| --- | --- | --- |
| pSB3C5\_Cl::Nb SLIC PCR | 274.6 | 1.849 |
| GBP3 SLIC PCR | 178.8 | 1.829 |

Chewbackk reactions of 3' ends using T4 DNA polymerase

| Reagent | Volume (µl) |
| --- | --- |
| 10x NEBuffer 2.1 | 2 |
| DNA fragments (~ 1000 ng) | 6 |
| MilliQ water | 10 |
| T4 DNA polymerase (0.3 U/µl) | 1 |
| DpnI (10 U/µl)/td> 1 | |

Incubate reactions for 30 minutes in a 37 °C waterbath

Add 2 µl of 10 mM dCTP to arrest the reaction. Inmediately transfer the tubes to an ice box

Assembly of DNA fragments

| Fragment | DNA size (bp) | Relative size | Equivalent DNA amount (ng) | [Chewback] (ng/µl) | Amount of chewback needed (µl) |
| --- | --- | --- | --- | --- | --- |
| pSB3C5\_Cl::Nb SLIC PCR | 3823 | 1 | 1000 | 50 | 18 |
| GBP3 PCR | 459 | 0.12 | 120 | 50 | 2.5 |

Mix the required DNA amounts in a microcentrifuge tube and incubate for 10 minutes at 37 °C in a waterbath

Clean up reaction using the BioBasic enzymatic protocol

Electroporate 100 ng of DNA into fresh electrocompetent E. coli BL21(DE3) gold

Previous
Next

Amplification of GFP (BBa\_E0040) USING oNb52 and oNb47

###### 2016/06/01

The GFP coding sequence was amplified from pSB2A1 using oNb52 (5' CCAGATCTGCGGCGGCAAGAATCATGCGTAAAGGAGAAGAACTTTTCACTGG 3') and oNb47 (5' GATTCTTGCCGCCGCAGATC 3')

| Reagent | Volume (µl) |
| --- | --- |
| 5x HF Phusion Buffer | 4 |
| Phusion (2U/µl) | 0.5 |
| dNTPs, 10 mM | 0.5 |
| oNb52, 10 µM | 1 |
| oNb47, 10 µM | 1 |
| pSB2A1 (10 ng) | 1 || MilliQ water | 12 |

Table 2. PCR cycle used for the amplification of different DNA fragments

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 15 | 30 |
| Annealing | X | 20 | 30 |
| Extension | 72 | 45 | 30 |
| Final extension | 72 | 300 | 1 |

A gradient PCR was done to amplify GFP. The thermocycler was set up in a way that each well had a different temperature (51 to 61°C)

The DNA fragments obtained were approximately 750 bp, the expected size of the GFP CDS

Previous
Next

Colony PCR of potential pSB3C5\_Cl::Nb\_GBP3 clones

###### 2016/06/02

The electroporation of the pSB3C5\_Cl::Nb\_GBP3 was succesful. Each plate had approximately 40 CFU.

Randome colonies were selected to set up colony PCRs using BioBrick suffix reverse and oNb51 as primers, ideally a DNA fragment of the size of the GBP3 gBlock should be observed.

\* Using a pipette tip, a little bit of the CFU was mixed with 50 µl of MilliQ water. Only 1 µl of the mixture was added to the PCR tube

| Reagent | Volume (µl) |
| --- | --- |
| 10x Pfu buffer | 2 |
| Pfu | 0.5 |
| dNTPs, 10 mM | 0.5 |
| oNb51, 10 µM | 1 |
| BioBrick suffix reverse, 10 µM | 1 |
| Colony dilution(\*) | 1 || MilliQ water | 14 |

Table 2. PCR cycle used for the amplification of different DNA fragments

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 120 | 1 |
| Denaturation | 98 | 30 | 30 |
| Annealing | 58 | 30 | 30 |
| Extension | 72 | 30 | 30 |
| Final extension | 72 | 300 | 1 |

The small fragment (~450 bp) belong to the GBP3 gBlock. Indicating that the construct was assembled

The colonies containing the DNA fragment were used to set up O/N cultures. The plasmid was isolated and sent for sequencing

The sequencing results determined that the GBP3 gBlock was not present in any of the plasmids. Repeat SLIC assembly

Previous
Next

Screening of potential pSBT3\_RNAPα::target clones

###### 2016/06/05

BglII digest of pSB1T3\_RNAPα::target clones to determine whether the contruct was assembled.

The RNAPα::target gBlock has a BglII unique cutting site on the 3' of its sequence.

The presence of a single cut indicates a positive clone.

| Reagent | Volume (µl) |
| --- | --- |
| BglII (ThermoFisher)/td> 1 | |
| Buffer O (ThermoFisher) | 2 |
| plasmid DNA (100 ng) | 5 |
| MilliQ | 12 |

Incubate for 1 hour at 37 °C

Agarose electrophoresis of results.

Clones 8, 9, 11, 12 cut once. The plasmids were sent for sequence analysis

The sequencing results showed that there is a missing region in the RNAPα::target gBlock in the 5' region.

Previous
Next

Single domain antibody library (sdAb) scaffold

###### 2016/06/25

Amplification of the sdAb scaffold via PCR

| Reagent | Volume (µl) |
| --- | --- |
| 2x Phusion MM, HF buffer | 50 |
| oNb45, 10 µM | 5 |
| BioBrick suffix reverse, 10 µM | 5 |
| pSB3C5\_Cl::Nb (Scaffold) gBlock(10 ng) | 1 |
| MilliQ water | 39 |

PCR cycle for the amplification of the sdAb scaffold

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 30 | 1 |
| Denaturation | 98 | 15 | 30 |
| Annealing | 68 | 15 | 30 |
| Extension | 72 | 30 | 30 |
| Final extension | 72 | 120 | 1 |

PCR worked well, however, several larger bands also appeared. They are likely multimers of PCR products as they occur in ~300 bp increments.

The multimers should not affect downstream digestion and cloning steps

![](https://static.igem.org/mediawiki/2016/d/d7/T--Lethbridge--NbScaffoldPCR.jpg)

Previous
Next

Restriction digest of sdAb scaffold

###### 2016/08/02

Double digest of sdAb scaffold PCR using NheI and BamHI

| Reagent | Volume (µl) |
| --- | --- |
| 10x Fast Digest buffer | 4 |
| sdAb scaffold PCR (103 ng/µl) | 30 |
| NheI Fast Digest (ThermoScientific) | 3 |
| BamHI Fast Digest (ThermoScientific) | 3 |

Incubate for 30 minutes at 37 °C

Spin column purification (BioBasic) and elute DNA in 40 µl MilliQ water

Set up NgoMIV difest using the eluted DNA

| Reagent | Volume (µl) |
| --- | --- |
| 10x CutSmart buffer | 10 |
| sdAb scaffold PCR (NheI and BamHI digested) | 40 |
| NgoMIV (10 U/µl) | 2 |
| MilliQ water | 48 |

Incubate for 30 minutes at 37 °C

Spin column purification (BioBasic) and elute DNA in 40 µl MilliQ water

The sample concentration is approximately 64 ng/µl

KpnI digestion of sdAb scaffold

| Reagent | Volume (µl) |
| --- | --- |
| 10x KpnI buffer | 10 |
| sdAb scaffold PCR (NheI, BamHI, NgoMIV digest) | 40 |
| KpnI (10 U/µl) | 2 |
| MilliQ water | 48 |

Incubate for 30 minutes at 37 °C

Spin column purification (BioBasic) and elute DNA in 40 µl MilliQ water

![](https://static.igem.org/mediawiki/2016/7/73/T--Lethbridge--RestrictionDigestofNbScaffold.jpg)

Previous
Next

Generation of CR1-2 library by oligo assembly and PCR

###### 2016/08/03

Oligo annealing of CDR1 and Framework region 2

| Reagent | Volume (µl) |
| --- | --- |
| oNb, 100 µM | 2 |
| oNb6, 100 µM | 2 |
| MilliQ water | 46 |

Heat oligo mixture to 95°C, then slowly cool on heat block (turn heat block off and let it reach room temperature)

Extend oligos using T4 DNA polymerase

| Reagent | Volume (µl) |
| --- | --- |
| Annealed oligos (4 µM) | 43 |
| 10x NEBuffer 2.1, | 5 |
| dNTPs, 10 mM | 1 |
| t4 DNA polymerase (3 U/µl) | 1 |

Incubate for 30 minutes at 12 °C for 15 minutes on thermocycler, then place on ice, clean up on spin column and elute in 40 µl of MilliQ water

Use 2 µl extended oligos as template for PCR (10 pmol)

PCR amplification of CDR1-2 oligos

| Reagent | Volume (µl) |
| --- | --- |
| 2x Phusion MM, HF buffer | 25 |
| Annealed/ Extended oligos (50 pmol) | 10 |
| oNb 7, 10 µM | 1 |
| MilliQ water | 14 |

| Step | Temperature (°C ) | Duration (s) | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98 | 120 | 1 |
| Denaturation | 98 | 20 | 5 |
| Annealing | 72 | 20 | 5 |
| Extension | 72 | 120 | 1 |

![](https://static.igem.org/mediawiki/2016/8/8b/T--Lethbridge--cdr12oligoassembly.jpg)

Generation of CDR3 libraries

###### 2016/08/03

Oligo annealing of various CDR3 DNA fragments

| Reagent | Volume (µl) |
| --- | --- |
| Forward oligo (oNb8, 9, 10, 11, 12), 100 µM | 2 |
| oNb13, 100 µM | 2 |
| MilliQ water | 46 |

Heat oligo mixture to 95°C, then slowly cool on heat block (turn heat block off and let it reach room temperature)

Extend oligos using T4 DNA polymerase

| Reagent | Volume (µl) |
| --- | --- |
| Annealed oligos (4 µM) | 43 |
| 10x NEBuffer 2.1, | 5 |
| dNTPs, 10 mM | 1 |
| t4 DNA polymerase (3 U/µl) | 1 |

Incubate for 30 minutes at 12 °C for 15 minutes on thermocycler, then place on ice, clean up on spin column and elute in 40 µl of MilliQ water

Phenol/chloroform extract DNA

![](https://static.igem.org/mediawiki/2016/1/1a/T--Lethbridge--cdr3library.jpg)\