Team:University of Sheffield /Protocols



=Protocols=

Making Agarose Gel
Standard 1%

Time:
 * ~1 hour

Required:
 * Gel tank, with combs, casting tray and relevant power supply
 * Agarose powder
 * TAE buffer, 1x
 * Ethidium bromide

Method

1.) The gel tank we normally use holds 150ml, therefore, take 150ml of 1x TAE Buffer

2.) The gel is supposed to be 10% agarose, therefore we need to add 1.5g agarose powder

3.) Combine the two in a chemical screw-cap bottle, and, leaving the lid slightly loose

4.) Cook on high heat in the microwave for 2 mins, or until ALL the agarose has dissolved (it may stick to the bottom, swirling may be required. *CAUTION* very hot liquid)

5.) If it starts to overboil, pause the microwave, allow to calm down, and continue.

6.) When all the agarose has dissolved, remove carefully! Its very hot! Oven gloves may be required.

7.) Add 10 µl of ethidium bromide per 100ml of buffer. Swirl to mix. Warning! Ethidium bromide is carcinogenic and therefore very dangerous!

8.) Leave until touchably cool before pouring into gel tank. Gently bathe bottle in cold water to speed up the process if necessary.

9.) Make sure combs are in place in tank, and casting tray is secured on all sides, then pour in the liquid.

10.) Leave until cool (~30 mins)

PCR Purification
...using QIAGEN Kit

Time:
 * ~1 hour

Required:
 * PCR Purification kit ideally. Instructions and reagents usually provided

Method

1.) Add 5 volumes of PBI buffer (provided) for every 1 volume of PCR product in an eppendorf tube. Make sure one eppendorf is large enough – you may need more.

2.) Put mixture in a QIAGEN column (purple) and into a 2ml collection tube (clear)(both provided). Remember, these columns only hold 700µl! Again you may need to repeat steps 3 and 4 more than once.

3.) Centrifuge at 10000-13000g for 30-60seconds

4.) ‘Flow through’ will be found in the collection tube: discard. The DNA has stuck to the column membrane. Repeat if you had more than 700µl of PCR+PBI buffer to start with

5.) The wash DNA of impurities further, add 0.75ml (750µl) of PE buffer (provided)(make sure the ethanol HAS been added to it! You have to do this yourself, but previous users of the kit may have done so and should have labelled the bottle so).

6.) Centrifuge for 1 min, and discard flow through.

7.) Centrifuge for a further 1min, as not all the PE buffer from the last step could fit in the collection tube.

8.) Discard flow through and put column into where you want your eluate (usually 1.5ml eppendorfs)

9.) To elute DNA, add 50µl of EB buffer (provided). This ‘unsticks’ the DNA allowing it to be washed through.

10.) Centrifuge for 1 min

11.) KEEP the flow through! This has your DNA in.

Making SOB Medium
Time:
 * ~20 mins, plus autoclaving time

Required:

EITHER


 * SOB powder, in which case make up as instructions

OR, per litre
 * 950ml dH20
 * 20g Tryptone
 * 5g Yeast Extract
 * 0.5g NaCl
 * 10ml of 250nM KCl
 * 5ml of 2M MgCl2
 * ~0.2ml 5M NaOH (may be required to adjust PH)

Method

1.) Combine dH2O, tryptone, yeats and NaCl, and shake until combined

2.) Add KCl

3.) Adjust to PH 7.0 with the NaOH

4.) Autoclave (20 mins liquid cycle)

5.) Add the MgCl2 just before use

Making SOC Medium
Method

Same as for SOB above, but add 20mM glucose

Sequential Ammalgamation of Wheatgerm Elements, C Albumin and Hydrogenated Fats
Whups! Not really sure how this was in my labbook. Ah well, put down your pipette and enjoy!



Time: ~1hour 30 mins

Required:
 * 110g caster sugar
 * 110g unsalted butter
 * 110g plain flour
 * 1 tspn baking powder
 * 1 tspn vanilla essence
 * 2 eggs
 * 1 tbsp milk

Method 1.) Beat sugar and butter together

2.) Add one egg and beat well, then add the second and beat well

3.) Add milk and mix

4.) Add flour and baking powder and mix

5.) Fill bun cases 2/3's full

6.) 12-15 mins at 190degrees celsius

7.) After cooling, ice as appropriate (eg our pic)

Making Cells Electrocompetant
by Dr Graham Stafford

Time:
 * ~ 3 hours cell growth
 * ~ 1hour 30mins execution

Required:
 * LB Medium
 * Ice cold dH20
 * Ice cold 1.5ml eppendorfs
 * Ice cold 10% glycerol

Method Centrifuge steps are 1000g for 15 mins

1.) Grow E.coli strain in LB until OD 600 = 0.4

2.) Centrifuge at 4 degrees C

3.) Pour of supernatant resuspend in 100 ml ice cold H2O

4.) Centrifuge at 4 degrees C

5.) Pour off supernatant, resuspend in 50 ml ice cold H2O

6.) Centrifuge at 4 degrees C

7.) Pour off supernatant, resuspend in 50ml ice cold 10% glycerol (sterile)

8.) Centrifuge at 4 degrees C

9.) Pour off supernatant, resuspend in 500ul ice cold 10% glycerol. Note: work only with freschly incubated cultures, as cultures old show no results

10.) Aliquot in 40ul portions into sterile, pre-chilled eppendorfs

11.) These can be stored at - 80 for months / years

Electroporation
Time:

Required:

Method

Making Cells Chemically Competant
Time:

Required:

Method

Tecan Fluorescence Measurement
Time:
 * Overnight prep
 * ~30 mins execution
 * 8 hours unsupervised measurement

Required:
 * TECAN machine
 * Overnights of cells to be measured
 * LB Medium
 * IPTG (in this case, to induce cells)

Method

1.) Grow 5ml of overnight cultures of DH5-alpha cells containing the plasmid with GFP-LVA.

2.) Resuspend the overnight cultures in 50 ml of LB medium until the OD600 reaches about 0.6. 3.) Quickly add 0.2 mM of IPTG to the medium and plate that into 96 well plate.

4.) To 96 well plate add 180 ul of LB + IPTG as a control, 180 ul of DH5-alpha cells not induced with IPTG, finally add 180 ul of DH5-alpha induced with IPTG. Carry out the aforementioned in duplicates.

5.) Use the high flow cytometry machine Tecan to measure the fluorescence every 15 minutes for 8 hours (excitation wv – 485 nm, emission wv- 535 nm )(including shaking = aeration of the cultures 2 minutes prior each measurement)

Our Usual PCR's
Time:
 * ~30 mins prep, depending on number of tubes
 * 1hour 20 mins running time

Required: OR
 * PCR kit
 * Taq Polymerase
 * 2x Taq Buffer
 * 10x MgCl2
 * DNTp's
 * Your DNA
 * Your Primers
 * dH2O

Method 1.) Set up 'Master Mixes': mixing everyhting together first, then aliquoting. Each reaction makes 20ul, and everything was added in the order written. n = number of tubes in reaction. For every reaction you need a blank and at least one duplicate. We usually had 3.

MM1,
 * Forward primer, 2ul x n
 * Reverse ptimer, 2ul x n
 * dH2O, 9ul x n

MM2, separate in case MgCl2 concentrations have to vary
 * dH2O, 5.7ul x n
 * DNTP's, 0.2ul x n
 * Buffer, 2ul x n
 * MgCl2, 2ul x n
 * Taq polymerase, 0.1ul x n

2.) Mix the two together, and aliquot 19ul into every tube

3.) Add 1ul of your DNA

4.) Put in machine.