THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 084 MOD: 1st Issue Page: 1 of 11

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1 Page: 1 of Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED Cloning of PCR products using blue/white selection HAZARDS 1. Chemical hazard Membrane binding solution HARMFUL Risk Phrases R20/21/22 - Harmful by inhalation, in contact with the skin and if Swallowed. R36/38 - Irritating to eyes and skin. 2. Chemical hazard IPTG HARMFUL 3. Sharp hazard scalpel blade 4. Exposure to UV light 5. Burns from hot heating block RISK ASSESSMENT 1. The risk of chemical exposure is low if Risk Controls are followed 2. The risk of cuts from scalpel blade is low if Risk Controls are followed. 3. The risk of exposure to UV light is low if the Risk Controls are followed. 4. The risk from burns from the heating block is low if Risk Controls are followed. RISK CONTROL 1. Wear appropriate PPE safety glasses, disposable gloves and laboratory gown. 2. Hold scalpel blade pointed away from hands and cut away from the body. 3. Dispose of scalpel blade immediately into Sharps container. 4. Wear face shield when using UV light. WRITTEN BY CHECKED BY AUTHORIZED BY NAME (signed) Rosa Baleato Ellen Byrnes Phil Dickson DATE 6 th October th October th October 2005 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of Place sign on heating block to indicate desired temperature, and warning other persons that heating block is in use. 6. Remove tube from heating block using tweezers. 7. Wear appropriate PPE safety glasses, disposable gloves and laboratory gown. 8. Training should be provided by personnel experienced in this procedure. 9. Training should be undertaken in General Laboratory Safety and Molecular Biology Safety. 2. Purpose: 2.1. To clone PCR products. Done by ligating PCR products into plasmid vectors of choice, then transforming into competent E.coli cells for propagation. 3. Equipment: 3.1. Submarine gel apparatus 3.2. UV light box 3.3. Vortex C Incubator or Heating block 3.5. Microfuge C Incubator or Heating block C Incubator C Shaking Incubator 4. Materials: ml tube % agarose gel (thick) 4.3. scalpel blade 4.4. sterile spreader ml falcon tubes 5. Reagents: 5.1. Promega s Wizard SV Gel and PCR purification kit (A9281) Membrane Binding Solution HARMFUL SV Minicolumn Membrane Wash Solution

3 Page: 3 of Nuclease-Free Water 5.2. Promega s pgem-t Easy Vector System (A1360) X Ligation buffer pgem-t Easy Ligase enzyme 5.3. LB media 5.4. DH5α strain competent cells (E.coli) 5.5. IPTG/X-gal agar plates for blue/white selection 5.6. X-gal 5.7. IPTG 5.8. LB +amp 6. Set Up: 6.1. Ensure that you have read and understood the Safety Precautions (Section 7 of this SOP) prior to commencing this procedure Use Promega s Wizard SV Gel and PCR purification kit, and pgem-t Easy (blue white selection) 6.3. NB: Cloning protocols are all the same regardless of which plasmid you use. 7. Safety Precautions: 7.1. Good laboratory techniques are to be used at all times. Method: 8. PCR Product Clean-Up 8.1. Run the PCR products of interest on a 0.8-1% gel (low agarose to make its removal easier). To maximize recovery, pour a thick gel and load as much of the product as possible. Leave empty lanes in between bands to allow for easy cutting of the gel slice Weigh a 1.5ml tube for each product, and record the weight of the empty tube Once gel has run far enough, place on UV light box and, wearing protection from the UV, cut out the bands of interest with a clean scalpel blade. Use a new blade for each band. Remove as much excess agarose from the gel slice as possible. Place into pre-weighed tubes Re-weigh tubes and calculate the weight of the gel fragment.

4 Page: 4 of From the Wizard SV purification kit, add 10 μl of Membrane Binding Solution per 10 mg of gel slice Vortex, and incubate at C for 10 minutes (or longer) to dissolve the gel fragment. Vortex the tube every few minutes to help dissolve Place one SV Minicolumn in a Collection Tube for each dissolved gel slice 8.8. Transfer the dissolved gel mixture to the column assembly and incubate for 1 minute at room temperature Centrifuge the column assembly in a microcentrifuge at 16,000 g (14,000rpm) for 1 minute. Remove the column from the Spin Column assembly and discard the liquid in the Collection Tube. Return the column to the Collection Tube Wash the column by adding 700µl of Membrane Wash Solution. Centrifuge the column assembly for 1 minute at 16,000 g (14,000rpm). Empty the Collection Tube as before and place the column back in the Collection Tube Repeat the wash with 500µl of Membrane Wash Solution and centrifuge the column assembly for 5 minutes at 16,000 g Remove the column assembly from the centrifuge, being careful not to wet the bottom of the column with the flow-through. Empty the Collection Tube and recentrifuge the column assembly for 1 minute to allow evaporation of any residual ethanol Carefully transfer the column to a clean 1.5ml microcentrifuge tube. Apply 25 µl of Nuclease-Free Water directly to the center of the column without touching the membrane with the pipette tip. Incubate at room temperature for 1 minute. Centrifuge for 1 minute at 16,000 g (14,000rpm) Discard the column and store the microcentrifuge tube containing the eluted DNA at 4 C or -20 C. 9. PCR Product Ligation into pgem-t Easy 9.1. To a clean 1.5ml tube, add: 3 μl purified DNA fragment 5 μl 2X Ligation buffer 1 μl pgem-t Easy 1μl Ligase enzyme 9.2. Leave at room temperature for 1 hr, or overnight (or over weekend) at 4 C.

5 Page: 5 of Bacterial Transformation (This should ideally be done in the afternoon so that bacteria can grow overnight, and not overgrow) Thaw E.coli competent cells on ice (DH5α strain is the most common). It is important to keep these cells on ice and treat them gently. Do not hold them in your hand to thaw out To a clean tube, add 5 μl of the ligation and place on ice. Pipette 50 μl of the competent cells into the tube containing the ligation Leave on ice for 30 minutes Heat-shock cells by placing at 42 C for 1 min, then placing immediately on ice for 5 minutes Add 150 μl LB media (WITHOUT AMPICILLIN!!!) to cells. Pipette up and down gently to mix, and then incubate at 37 C for 1 hour During this hour, prepare the IPTG/X-gal agar plates for blue/white selection. You will need two plates per DNA fragment. Coat LB Agar ampicillin plates (100 μg/ml amp) with X-gal and IPTG. To do this, pipette 100 μl of 100 mm IPTG, and 20 μl of 50 mg/ml X-Gal onto each plate and spread using a sterile spreader. Sit plates with lids on at RT for 5-10 minutes to allow plates to soak up reagents. Then leave plates inverted at room-temp, or place back at 4 C Once the 1 hr incubation is up, pipette a 10-1 (1:10) and 10-2 (1:100) dilutions of your bacteria (in a total volume of 50 or 100 μl of LB) into the centre of you agar plates, and spread evenly using a sterile spreader Leave for 5 10 mins for liquid to be absorbed, then invert plates and incubate overnight at 37 C In morning, remove plates from incubator and circle any white colonies. Wrap in cling-wrap and place plates in the fridge for a few hours to ensure no colour change of white colonies. Plates can be left in the fridge for up to two weeks, but it is best to work with them ASAP. NB: White colonies indicate successful ligation of the DNA insert into pgem-t Easy and the disruption of the β-galactosidase gene flanking the multiple cloning site. Blue colonies suggest the plasmid re-ligated without the insert, and hence can still produce a functional β-gal enzyme, resulting in the blue precipitate which is formed when β -gal metabolises

6 Page: 6 of 11 the X-gal. However this is not always fool-proof, and hence we have to check that any white colonies do contain our insert. 11. Mini-prep Plasmid Isolation and Restriction Enzyme Digestion Prepare a 50 ml falcon tube for each colony that you want to examine. Usually, choose 2 or 3 colonies for each fragment (i.e. make 3 cultures for each gene you are trying to clone). Place 5 ml LB +amp in each tube, under sterile conditions Using a sterile inoculation loop, or a sterile yellow pipette tip, pick the colony of interest and inoculate the 5 ml of media. Be sure to pick a good-sized, freestanding white colony Screw lid on firmly, and place in shaking incubator overnight at 37 C, shaking at rpm. Do this relatively late in the afternoon to ensure that cultures do not overgrow, as this can minimize plasmid yields In the morning, perform minipreps on 1-2 ml media using plasmid miniprep kits according to manufacturer s instructions. NB1: Usually, the preps involve an elution step to elute DNA off a column. These kits provide an elution buffer, which is basically TE buffer. It is best to avoid using these buffers and elute in DNAse-free water, as the EDTA in these buffers can interfere with down-stream applications. Also, the kit instructions usually recommend eluting in large volumes, resulting in very dilute preps. It is usually best to use a half or even a quarter of the volume of what they recommend (depending on the kit and column type). NB2: pgem-t Easy is a high copy plasmid, so you should not need to prep any more than 2 ml of culture. Miniprep kits come with DNA-binding columns, and it is possible to overload these columns if too much culture is prep d Once mini-preps have been carried out, quantitate plasmids on the spec. a 1:100 or a 1:50 dilution should suffice Restriction enzyme digestion: Digest anywhere from 200ng 1 μg plasmid (depending on yields don t want to use it all up yet) To a clean 1.5 ml tube, add: 200 ng 1 μg plasmid

7 Page: 7 of 11 Buffer to 1X (buffers are usually a 10X buffer) 1 μl restriction enzyme Water to a final volume* * final volumes are depended on the concentration of your plasmid. 10 μl to 30 μl digestion reactions are usually easiest to work with. Try and use the smallest volume possible Incubate for hrs at 37 C Run half of digest on a 1 1.5% agarose gel to analyse. If possible, run a small amount of undigested plasmid alongside digested samples for comparison If the ligation and transformation has been successful, a band the size of your insert (and the bases on either side depending on where the restriction enzyme recognizes the DNA) should be visible, as well as the digested plasmid. 12. DNA Sequencing In order to confirm the sequence of the inserts, it is necessary to sequence the plasmid. Choose which samples to clone based on restriction enzyme digest results. Sequencing is done at the AGRF (Brisbane) and samples are sent through express post. Overall it should take 5 days from sending to receive results. When using pgem-t Easy, inserts are sequenced using T7 and SP6 primers (primers that bind to the T7 and SP6 promoter sites on either side of the MCS we have these primers in the lab). The following instructions were obtained from the AGRF website: How to Prepare Your Purified DNA (PD) Samples For this service the AGRF is offering the DNA labelling (sequencing) reaction and cleanup followed by the analysis of the samples on a AB3730xl sequencing platform. The results are sent to a secure FTP site in the form a chromatogram and text file of the sequence data. Turnaround for this service is between 3 5 working days after your samples have been received. * (1) IMPORTANT: The two most important factors in florescent DNA sequencing are the quality and quantity of DNA template.

8 Page: 8 of 11 (2) Template Quality: Automated sequencing on our platforms (3730xl) is much more sensitive to many contaminates compared to older capillary systems (3700), gel based platforms and PCR. Contaminates include RNA, protein, carbohydrates, lipid and common buffer salts. To ensure quality, templates should be analysed by both spectrophotometer (OD260/OD280 = 1.8 to 2.0) and gel electrophoresis with a known mass standard. (3) Template Quantity: It is very important to know how much DNA template you are using in order to ensure reliable, reproducible results. The quantity of DNA template needed will vary with its size, which is outlined below (Table 1). Please remember that the template amounts outlined are only guides, optimisation will be needed! We prefer template DNA to be quantitated by gel electrophoresis with a known mass standard as we have found that spectrophotometry commonly overestimates concentration. (4) For clients using our Purified DNA service please submit your template and primer premixed in a TOTAL VOLUME of at least 8μL. Samples will only be accepted in 1.5mL flip-top microcentrifuge tubes. You may wish to send 10-12uL to account for loss of sample due to pipetting and evaporation. The suggested amounts of primer and template in 8uL are as follows: PRIMER 6.4 pmol TEMPLATE: PCR Product ~2-6 ng per 100 base pair Plasmid, Single-stranded ng Plasmid, Double-stranded ng (5) Sample Names: Please keep sample names simple (8 characters long). Clearly label your 1.5 ml tubes both on the lid and on the side. If there is room you may wish to number your samples in the order that you submitted them. (10) Resuspending DNA Template As with plasmids, PCR fragments should be resuspended in sterile distilled water or a buffer that does not contain EDTA. Since EDTA chelates Mg (Mg2 + being a cofactor for

9 Page: 9 of 11 the DNA polymerase) the presence of EDTA in the cycle sequencing reaction can dramatically inhibit the reaction efficiency. (11) Sequencing Primers Sequencing Primers should be selected to have one or more Gs or Cs at the 3 -end. The length of the primer should be between 18 and 30 bases, with optimal being bases. Desirable G-C content is 40-60% with a Tm around 55C. Design your primer about bases before the start of your sequence of interest because the first bases of sequence are usually messy and unreliable

10 Page: 10 of 11 pgem-t Easy Multiple Cloning Site

11 Page: 11 of Shutdown: Change History: Issue Number: 1st Issue Date Issued: 1 st November Issue Number: Date Issued: Reason for Change: