Protocols

AGAROSE GELS	DNA EXTRACTION
RNA EXTRACTION	RT REACTION
PCR REACTION	GEL EXTRACTION
LIGATION	TRANSFORMATION

MINI-CULTURES	MINI-PREPS (QIAGEN)

Agarose Gel

Measure the amount of Agarose needed. Deposit into a 50 ml Erlenemyer flask. Add the corresponding amount of TAE buffer. Microwave to boiling point until completely dissolved ( watch when so it does not overflow during boiling). Take out of microwave and cool slightly and add corresponding amount of SyberSafe. Swirl to mix and poor into gelcaster. Make sure the comb is in place ! Immediately wash out Erlenmeyer flask with hot water and clean.

A normal gel base unit with 8 well comb holds 25 ml and gives wells that can hold ~ 22 ul sample. (if you want to load more sample per well, adjust the total gel volume to 30 ml )

The longer rectangular size holds 40 ml. ( this base can hold two rows of combs for a total of 16 samples)

The larger your expected DNA bands, the lower the % of Agarose to use. The smaller your DNA bands, the higher the % agarose to use. In general, I like to use a 1.5 % gel. For bands below 300 bp, a 2 % gel gives better separation. For bands above 1000 bp, use a 1% gel.

Quantities of Agarose needed for a normal gel ( 25 ml)

1.0 % gel : 0.25 gr ; 1.5 % gel : 0.375 gr ; 2.0 % gel : 0.50 gr

Quantities of Agarose needed for an extended gel ( 40 ml)

1.0 % gel : 0.40 gr ; 1.5 % gel : 0.60 gr ; 2.0 % gel : 0.80 gr

Amount of SyberSafe = 1 ul per 10 ml of gel.

RNA Extraction

RNA extraction usually provides us a pool of all RNA. It includes all the mRNA the cell has at the moment of cell preservation.

For medium sized oligochaete worms, 6-10 worms is usually sufficient or 50-70 mg of N2-frozen, ground tissue.

Trizol Method :
- Add 1 ml Trizol to tissue and ground with tissue grinder (glass or plastic hand homogenizer)
- Transfer to eppendorf tube, let sit for few minutes at Room Temp. and add 200 ul Chloroform ; vortex well
- Centrifuge for 10 min, max. speed in table centrifuge
- Remove top layer with 200 ul micropipette ( Remove about 450 ul into new eppendorff tube and avoid taking any of the middle white layer with it)
- Add 500 ul icecold Isopropanol; vortex well
- Centrifuge for 10 min at max speed
- Remove supernatant, keep pellet and add 70% cold Ethanol--centrifuge for 5 min at 7000 g.
- Remove supernatant and air dry pellet
- Dissolve pellet in nf water ( 30-70 ul, estimating on pellet size)
- Measure concentration of RNA on 2-3 ul into 50 ul TE buffer ( 260/280 should be > 1.9)

The other method used is the RNA GenElute kit from Sigma ! It works well with smaller samples (<50 mg). Anything over that cloggs up the membrane.

Sigma GenElute Method :
- Make 1 ml of lysis buffer (good for 2 RNA extractions) : add 10 ul 2-MCE to 1 ml lysis solution
- Add tissue to 500 ul Lysis solution and grind with plastic grinder in eppendorf tube
- Transfer to blue ringed filtration column and spin down (30 sec)
- Save filtrate and add equal volume 70% EtOH ( 500 ul)
- Mix well and transfer to clear binding column; spin through (30sec)
- Discard flow through and repeat if needed with remaining solution
- Wash with 500 ul Wash Solution 1
- Wash 2 times with 500 ul Wash Solution 2
- Spin empty for 1 min. to remove EtOH
- Transfer column to empty column and elute RNA with ~ 50 ul nuclease free elution solution.
- Measure concentration of RNA on 2-3 ul into 50 ul TE buffer (260/280 should be > 1.9)

DNA Extraction

I perform DNA extraction either with a Promega Wizard kit or with a Sigma GenElute kit.

Promega Method :
- To be updated

The other method used is the DNA GenElute kit from Sigma !

Sigma GenElute Method :
- To be updated

RT Procedure

Once we have RNA, we want to convert the mRNA pool into more stable DNA ( called the cDNA pool).

This is done by using Reverse Transcriptase and taking advantage of the Poly-A tail of mRNA's.

MMLV RT procedure ( Promega) :
- In a PCR tube, add 1 ug RNA, 1 ul OligodT15 primer and enough nf H20 to make 13.5 ul.
- Mix and incubate at 70 C in PCR cycler for 5 minutes, 50 C for 2 min, 35 for 2 minutes
- Take out and ice for a few minutes
- Add 4 ul 5x Buffer, 1 ul MMLV enzyme, 1ul dNTPs and 0.5 ul RNase
- Mix and incubate for 60 minutes at 42 C, followed by 15 minutes at 70.
- Put on ice and add 80 ul PCR water.

This cDNA is now ready for PCR reactions ( usually 2-3 ul per PCR reaction).

PCR Reaction

We assume that we do a PCR on 3 ul sample. If for example you need to do a PCR on 5 ul sample, lower the nf-water by 2 ul. We also assume that Left Primer and Right primer are the same for all PCR reactions.

For 'n' PCR reactions, mix the following in a PCR tube
- 12.5 ul Mastermix times n
- 1.5 ul 25 mM MgCl2 times n
- 6 ul nf-water times n
- 1 ul Left Primer times n
- 1 ul Right primer times n
Mix by pipetting and dispense 22 ul into n PCR tunes
To each PCR tube add 3 ul of your sample to be amplified
QuickSpin in microfuge to mix and place samples in PCR machine ( PCR machine should be ready to go and paused at 92 C for HotStart)

Assume you have different primers for each reaction but using the same sample source, in that case you can proceed as follow:

For 'n' PCR reactions, mix the following in a PCR tube
- 12.5 ul Mastermix times n
- 1.5 ul 25 mM MgCl2 times n
- 6 ul nf-water times n
- 3 ul sample source times n
Mix by pipetting and dispense 23 ul into n PCR tunes
To each PCR tube add 1 ul of your specific Left and Right primer resp.
QuickSpin in microfuge to mix and place samples in PCR machine ( PCR machine should be ready to go and paused at 92 C for HotStart)

Basic PCR conditions

94 C for 2 min
Cycle between the following steps
- 94 C for 50 sec (=denaturation step)
- 55 C for 50 sec (= annealing step for primers to latch on)
- 72 C for 50 sec (=time for polymerase to anneal and extend the new strand)
- repeat this for 38 cycles
Final Extension at 72 C for 5 minutes
Cool at 12 C for infinity (but remove ASAP for gel electrophoresis)

Rule of thumb is 1 minute extension for every 1 kB of DNA length. So if your expected band size is 250 bp, you can easily decrease Annealing and Extension to 30 seconds.

Gel Extraction

Extract amplicon (band) of interest with razorblade or gelplug extractor
Put into eppendorf tube and freeze - or add 300 ul Qiagen buffer QG per 100 mg agarose plug
Heat for 10 minutes at 55 C
Add 100 ul Isopropanol per 300 ul buffer QG added; vortex
Spin through Gel extraction column (30 sec), discard flow through and spin wash (30 sec) with 500 ul QG buffer
Spin Wash again with 700 ul PE buffer (30 sec) and then spin for 1 min. without any fluids on top to dry the membrane
Elute with 20-25 ul EB buffer, warmed to 65 C. If desired, put the flow through back over the column and spin again to get maximum elution
NOTE : the higher the amount of EB used, the more recovery but the less the concentration. The volume used above seems to work well for Ligation procedures.

Ligation with pGEM-T Easy

For 1 ligation, make a mix of the following (adjust accordingly when doing 'n' ligation)
- 2.5 ul Ligation buffer (2x)
- 0.5 ul pGEM vector
- 0.5 ul Ligase
- 0.5 ul nf-water
- Add 1 ul of your DNA to be ligated
Keep at RT for 2 hrs or overnight in fridge

Transformation with TOP-10 cells

Unfreeze TOP10 cells (1 tube = 50 ul) slowly in bucket of ice
If doing 2 transformations, put an eppendorf tube with a yellow pipette tip in ice to cool down
After about 30 minutes, transfer 25 ul top 10 cells with cold pipette tip into cold eppendorf tube ( do it fast to prevent warming of cells)
To each tube add 1 ul of your ligated DNA
Put on ice for 2 minutes and then heatshock for 35 seconds and imediately put back on ice for 2 minutes
Add 125 ul LB media to each tube and put at 37 C for at least 1 hr
In mean time, warm up two LB/Amp plates and spray surface with IPTG/Xgal ; put at 37 C
Plate each plate with 70-90 ul of transformed cells
Incubate overnight at 37 C.
White colonies are the ones that have the plasmid with insert.
NOTE : a single tube of TOP10 cells can be divided into 3 to perform 3 transformations. All depends on your technique how succesful this will be. So, in this case, 1 ul of ligated vector will be added to ~17.5 ul TOP cells.

Dr. C. Doumen - 2010