cDNA/AFLP Protocol

 

Preparation of Para-magnetic beads from Promega cat#Z5482:

a) suspend magnetic particles in bottle - transfer 200 ul (200 ug) of beads per sample

of RNA to be purified to a microfuge tube or tubes.

b) place microfuge tube in magnetic stand for 30 sec or longer to collect the beads.

c) remove supernatent and wash 3X with 200 ul of 0.5X SSC

d) finally resuspend the SA-PMPs in 100ul of 0.5X SSC for each 200 ul of beads originally removed - each 100 ul aliquot should be in its own labelled microfuge tube, 1 for each RNA sample.

note: this should not be done more than 30 mins before the beads are to be used.

 

Hybridization of biotinylated polyT primer:

a) add H2O to 20 ug - 200ug (more is better) of RNA to make a final volume of 500 ul

b) heat to 65C for 10 mins.

c) add 1.5 ul of 100 uM poly d(T)25V and 13 ul of 20X SSC

d) let cool to room temperature (~10min)

e) add RNA/poly T mix to the previously prepared 100 ul of SA-PMPs; mix gently and let sit at room temperature for 10min

f) capture PMPs with magnetic stand - carefully remove supernatent and wash 3X with 300 ul of 0.1X SSC.

g) wash w/ 50 ul of 1X reverse transcriptase buffer.

 

1rst Strand cDNA synthesis:

a) aliquot samples of Reverse Transcriptase Mix:

• 13 ul H2O
• 2ul 10X Stratagene rxn buffer
• 2ul 0.1M DTT
• 1ul 10mM dNTP mix
• 1ul RNase Inhibitor (Gibco/BRL)
• add to polyA-containing beads

b) heat mix to 37C for 2 mins - then add 1 ul (50U) of MMLV reverse transcriptase (Stratagene).

c) incubate at 37C for 15 mins, then 42C for 45mins, mixing every 15mins

2 nd Strand synthesis:

a) add 140 ul of 2 nd strand cDNA rxn mix to 1rst strand synthesis rxn:

• 119 ul H2O
• 16 ul 10X E. coli DNA ligase buffer
• 3 ul 10 mM dNTP mix
• 1 ul RNase H (1 U)
• 1 ul E.coli DNA pol I (10 U/ul)
• 0.25 ul E.coli DNA ligase (10 U/ul)
• 140 ul

b) incubate at 16C for 2hrs.

c) collect beads with magnetic stand, and remove supernatent.

d) wash once in 1X NEB TaqI buffer

e) resuspend in 40 ul of 1X NEB TaqI buffer (w/1X BSA - 100X stock sent w/ enzyme )

Digestion:

a) to 40 ul of cDNA add: 1 ul (10U) of Taq I.

b) digest at 65C for 2hrs

c) to digest w/ NcoI, add: 1ul Nco I (10U/ul)

d) digest at 37C for 2 hours

e) collect PMPs with magnetic stand, and remove the 40 ul of supernatent (Ligation template) to a new tube. The digestion should liberate the cDNA from the beads.

Preparation of Adaptors

a) for Taq I adaptors - make a 45 uM stock solution of adaptors - to 11 ul of 10X Gibco/BRL buffer 3 add 50 ul of the top strand adaptor (100 uM) to 50 ul of the bottom strand adaptor (100 uM).

b) for Nco I adaptors - make a 5 uM stock solution of adaptors - add 5 ul of the top strand adaptor (100 uM) and 5 ul of the bottom strand adaptor (100 uM) to 80 ul H2O + 10 ul 10X BRL buffer 3.

c) heat both to boiling, and let cool to RT.

 

Ligation of Adaptors:

a) to the 40 ul of digested cDNA add:

• 1 ul Taq I adaptor (45uM)
• 1 ul Nco I adaptor (5uM)
• 2.5 ul 10 mM ATP
• 0.5 ul NEB T4 DNA ligase buffer
• 0.2 ul T4 DNA ligase
• 45 ul total

b) 37C for 3hrs or ON @ 30C

PCR/preamplification

a) mix reaction on ice:

• 15 ul H2O
• 2 ul PCR buffer (Gibco/BRL)
• 0.6 ul 50 mM MgCl2
• 0.5 ul 10mM dNTPs
• 0.5 ul Taq I primer (10 uM)
• 0.5 ul Nco I primer (10 uM)
• 0.25 ul Taq DNA pol
• 1 ul template DNA
•  

b) PCR reaction

1. 94C 2min

2. 94C 30sec

3. 50C 30sec

4. 72C 1 min

5. goto 2, 20X

run an 8 ul aliquot on a 2% gel, then dilute 10X to 20X with water, taking care to equalize any concentration differences in the samples.

Primer Labelling

a) make reaction mix:

• 24 ul H2O
• 10 ul Nco I primer (10 uM)
• 10 ul 33P-ATP
• 5 ul 10X PNK buffer
• 1 ul T4 polynucleotide kinase (10 U/ul)

you can also 1/2 the amount of 33P in this reaction and still get useful primer

b) incubate at 37C for 30 mins

To Label AFLP ladder (from Gibco/BRL)

a) mix:

• 2 ul H2O
• 1 ul 10X rxn buffer (NEB)
• 4 ul AFLP ladder
• 2 ul 33P-ATP
• 1 ul T4 Polynucleotide kinase

b) incubate at 37C for 10 mins

c) before loading, heat to 70C for 5 mins

PCR

AFLP cocktail (/reaction)

• 1 ul preamplified DNA
• 0.5 ul labelled NcoI adaptor
• 0.6 ul unlabelled Taq I adaptor (10 uM)
• 0.4 ul 10mM dNTPs
• 2 ul PCR buffer
• 0.6 ul 50 mM MgCl2
• 0.4 ul Taq DNA polymerase
• 14.5 ul H2O

~undefinedthis rxn can be scaled down to a 10ul.

PCR program:

1. 94C 2min

2. 94C 30 sec

3. 65C 30 sec, -0.7C per cycle starting next cycle

4 72C 1min

5 goto 2 11 times

6 94C 30sec

7 56C 30sec

8 72C 1 min

9 goto 6, 25X

10. 72C 2min

11 4C hold

 

After PCR add an equal volume of 2X loading buffer:

 

0.1% Xylene cyanol

0.1% Bromophenol blue

in Formamide.

 

To reduce volume and increase the concentration of your products, you may heat your samples at 90C for 30 mins to evaporate some of the sample.

Denaturing Acrylamide Gel Electrophoresis

For BioRad Sequigen GT NA Electrophoresis cell

Set up apparatus, make 150 mls of Acrylamide Soln:

 

• 63 g Urea
• 15 ml 10X TBE
• 19 mls 40% Acrylamide Solundefined
• H20 to 150 mls
• add 150 ul fresh 25% APS
• 150 ul TEMED

 

~undefined 40% Acrylamide; 19:1 Acrylamide:Bis-Acrylamide

-use gloves, lab coat, and dust mask when making - Acrylamide is a neurotoxin!

After adding APS and TEMED, mix thoroughly and take soln' up slowly in syringe. After evacuating any air bubbles, slowly inject solution into loading apparatus, which should be on a slight incline. After you have filled the space, level the plates and insert the comb.

Allow to polymerize 1 hr.

 

Add buffer (1X TBE) to top chamber - to 1 cm from top of rig. Add 1X TBE to bottom chamber - being careful not to fill with more than 500 mls of buffer, yet ensuring that the bottom of the glass plates are submerged.

 

Run gel on TEMPERATURE mode - 45C. Set for 80 W. Pre-run, until gel temperature is 45C. Pre warming buffer in microwave can greatly decrease amount of time needed for the pre-run (greater than 1 hr if starting from RT).

 

Once at 45 C, turn off power supply, and load samples. Our wells will take less than 7 ul of sample.

 

Once all the samples are loaded run until fast blue (Bromophenol blue) dye front is off of the gel. Stop power supply and drain top buffer resevoir with hose and valve on IPC unit. Pour the rest of the top buffer into the sink. Remove gel to a peice of bench coat, to soak up any of the lower buffer from the bottom of the gel.

 

To remove gel, take off clamps, and carefully peel the long plate off of the IPC unit - hopefully the gel will stick to the IPC plate only. Once this is done, mist the gel with water, and carefully place a piece of Wattmans blotting paper on the gel. Carefully peel the filter paper off, ensuring that the gel is sticking uniformly to the blotting paper. Once off, dry at 80 C for 1-2 hrs.

 

At this point remember to dispose of the lower (radioactive) buffer. Pour carefully into the 33P waste container using the funelled corner of the apparatus' base.

 

After drying, Expose! (ON - 3 days)