DsRNA-RT-PCR-Southern-Seq-analysis-from-Sarah

(To make 50 ml: 25 ml of formamide, 12.5ml of 20XSSC, 0.05 g of N-aurolsarcosin, 100 ul of 10% SDS, 10 ml of 10%blocking solution and add water up to 50ml)

Low stringency wash buffer

2X SSC , 0.1% SDS
(50 ml 20X SSC + 5 ml 10% SDS add dH2O to 500 ml)

High stringency wash buffer

0.5X or 0.1 X SSC, 0.1X SDS
(12.5 ml or 2.5 ml 20X SSC + 5ml 10% SDS, add dH2O to 500 ml)

Solution for Dig Detection

Maleic acid buffer (0.1 M maleic acid (11.6g/Lin molecular cabinet), 0.15 M NaCl (8.766g/L), pH 7.5 with solid NaOH, autoclave keep in room T)
10% blocking solution (10g block reagent (on agrose shelf) /100ml dd H2O, keep in-20C)
Blocking solution (45 ml maleic acid buffer +5 ml 10% blocking solution
Washing buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5, 0.3% Tween)

make it before use: 100 ml Maleic acid buffer + 300 ul (cut blue tip) Tween 20 (keep in glass door shelf)

Antibody solution (1ul anti-Digoxigenin-AP (keep in 4C white frig.) +10 ml Blocking solution)
Detection buffer (0.1M Tris-HCl, 0.1 M NaCl, pH 9.5, keep in room T.)

1.5760 g Tris + 0.584 g NaCl in 100 dd H2O or 7.88 g + 2.92 g in 500 ml

Others need to prepare:

Boiling water, 37 C incubator (soak 37 at PCR machine), shaker, plastic bag, 2 mm Whatman paper, paper towel, plastic tray, glass tray, plastic wrap. UV stratalink (MBB 7161), Film (Kadar), Film developer m (MBB next to UV room)

Method

Day 1

1. Transfer RNA to membrane

Run 1% agrose gel with 0.5 TBE buffer at 60-70 V (dsRNA do not need formdehyde gel), take a picture.
Put gel in a glass tray and add depurinate solution 100-200 ml to cover the gel and put the glass tray on shaker (beside the sink) for 10 min. at room T.
Denature the gel (RNA) in denature solution for 20 min.
Neutralization solution for 20 min.
Rinse with 10 X SSC
Set up Nothern transfer (upside down electoforesis tray in a plastic box ), put 2 Whatman paper, drop 10X SSC , put gel on the paper, cut positive charge membrane same size as gel and put on the gel, drop 10 X SSC on the membrane and drive out the bubble. Put 2 Whatman paper and add 10-20 cm paper towel, add heavy box on the top.
Transfer overnight at room T or three hours.

2. DNA Dig labeling

- Prepare boiling water and 37C (soak 37 file in PCR, or 37 incubator)
- Denature 15 ul DNA template (PCR tube) in boiling water for 10 min and quickly chill in ice.
- Add: 2 ul Hexanucleotixde mixture (!0X)

2 ul dNTP dig Labelling mixture (10X) (order from Roche)

1 ul Klenow enzyme.

4) Incubate the reaction at 37C for overnight (soak 37 file in PCR)

Day 2

1.Transfer RNA to membrane

UV stratalink RNA membrane at 1200 nm for 10 seconds. (MBB Like a microwave, door is not closed properly, need left up a little. Put membrane on a tower paper and put in. set 1200. wait for finish)
Bake 1 hr at 80C (optional)
Prehybridization

- Pre-warm hybridization solution at 42C (68C)
- Add 10-15 ml /100 cm²membrane in bag and sealed bag without bubble (green light off on the seal machine means seal ok).
- Shaking bag (put between rack in the 42C water bath) for 2-4 hours at 42C. (If film show lot of background then increase the temperature to 55C or 68 C)

Hybridization

Add total labeling DNA (50ul or 30ul) into 3.5 ml hyb solution.
Shaking at 42C overnight.

2. DNA Dig labeling

5) Add 2 ul of 200 mM EDTA

6) Add 1 ul of Glycogen solution (optional)

7) Precipitate the probe with 2 ul of 4 M LiCl and 60 ul of 95% Ethanol and incubate at -80 for 1 hr.

8) Centrifuge 12,000 x g for 15 min.

Wash with 70% Ethanol and centrifuge for 5 min

Dry the pellet and resuspend in 30 or 50 ul of TE buffer and store at -20.

Day 3

1. Wash with low stringency wash buffer

Cut a corner of the hyb bag and pour Hyb solution into a tube and keep it at -20, it can be use again.
Wash membrane 2x in plastic box at room T by shaking for 15 min each time (if lot of background, wash longer)

2. Wash with high stringency wash buffer

Wash membrane 2x in plastic box at room T by shaking for 15 min (if lot of background, wash at 68C, preheat high buffer at 68C but it will give you a faint band. So first you need to see band and then adjust background)

3. Dig Detaction

Pre-warm concentrated CSPD (substrate) in dark drawer (light sensitive) from 4C frig.
Take out membrane and wash plastic box with tap water and dry it with paper towel.
Add wash buffer and membrane, shaking 2 min at room T.
Discard wash buffer and wash box and dry it again.
Add Blocking solution to box, shaking at room T for 30-1 hr. Discard blocking solution Add 10 ml Antibody solution, shaking 30 min at room T.
Wash membrane 2 x 15 min with sashing buffer.
Equilibrate membrane 3 min in detection buffer.
Clean a plastic development folder with water and dry with tissue paper.
Place membrane (RNA/DNA face up) in the middle of folder and add 1 ml CSPD (10ul concentrated CSPD + 900ul detection buffer) on the membrane.
Cover the other side of folder, it will spread substrate (CSPD) evenly on the membrane (do not let air bubble form between membrane and folder)
Incubate at room T for 5 min. prepare expose box and film in the dark room.
Put folder in the box and put a frame in the box and turn off the light

4. Expose the sealed membrane to film

In the dark open the film box, inside has another paper bag (open side is opposite with the film box open side), take one film put it into expose box within the frame, take out the frame and close the expose box. Put back the film paper bag back to the film box (open side need opposite).
Turn the light, put expose box in the drawer to expose for 2 hrs.
Develop the film at MBB develop room (next to the UV room) between 9:30 -5:30.
Turn off the light and take out the film put on the left side of the develop machine and move the film into the machine until hearing the peep and turn on the light.
Waite for about 3 min the film will come out in another side of the machine

Protocol 1-2 Sarah Chen (April 6, 2006)

Northern Blot for dsRNA with 32-p labeling

Reagents

RNA Treat dsRNA with CF11, with RQ1 and RNase, resuspend in dd water (all solutions made with ddH2O)

DNA Clone’s DNA fragment

Solutions for Northern blot:

0.5 X TBE Buffer

Depurinate solution (0.2N HCl)

(1ml 12N HCl +60ml dd H2O or 10 ml 1N HCl + 50 ml dd H2O

Denature solution (50mM NaOH, 1.5 M NaCl)

(1g NaOH + 43.8g NaCl in 500 ml ddH2O)

Neutralization solution (1 M Tris, 1.5 M NaCl, pH 7.4)

(60.57 g Tris- HCl + 43.8 g NaCl in 500 ml)

20X SSC (3M NaCl, 0.3 M sodium citrate, pH 7.0 (20C) and autoclave.

(87.66g NaCl + 44.115g sodium citrate in 500 ml, pH 7.0)

Solution for Labelling:

Use promega Prime-a-gene Labeling system (50 ng DNA)

Solution for pre-Hybridization and Hybridization:

Use Sigma H7033-125ML PerferctHyb-plus Hybridization buffer for both pre-hyb and hyb

Wash 1 Low stringency wash buffer

2X SSPE , 0.5% SDS
(108 ml 15X SSPE + 20 ml 20% SDS add dH2O to 800 ml)

Wash 2 High stringency wash buffer

0.1 X SSPE, 0.5% SDS
(5.3 ml 15X SSPE + 20 ml 20% SDS, add dH2O TO 800 ml)

Others need to prepare:

Boiling water, 2 mm Whatman paper, paper towel, plastic tray, glass tray, plastic wrap. UV stratalink (MBB 7167), Hyb-tube (small and big), 32-p (order each Wed. from Amersham), MicroSpin column from Amersham), Cassette, Film (Kadar), Film developer (MBB next to UV room), Hybond-N + membrane (Amersham).

Method

Day 1

1. Transfer RNA to membrane

Run 1% agrose gel with 0.5 TBE buffer at 60-70 V (dsRNA do not need formdehyde gel), take a picture.
Cut the left bottom of gel.
Put gel in a glass tray and add depurinate solution 100-200 ml to cover the gel and put the glass tray on shaker (or hand shake) for 10 min. at room T. (rinse with d-water)
Denature the gel (RNA) in denature solution for 20 min, rinse with d-water).
Neutralization solution for 20 min.
Rinse with 10 X SSC
Set up Nothern transfer (upside down electoforesis tray in a plastic box ), put 2 Whatman paper, drop 10X SSC , put gel on the paper upside down, cut positive charge membrane same size as gel ( cut the left bottom of the membrane too) and put on the gel, drop 10 X SSC on the membrane and drive out the bubble. Put 2-4 Whatman paper and add 10-20 cm paper towel, add heavy box on the top.
Transfer overnight at room T or three hours.

Day 2

1.Transfer RNA to membrane

Turn the hyb-chamber and set the temperature at 65 or 68 C for bake.

UV stratalink RNA membrane at 1200 nm for 10 seconds (wait to 0 nm) 1or 2x at MBB room 7167. It like a microwave, door is not closed properly, need left up a little. Put membrane on a tower paper (dsRNA face up) and put in. set 1200. start and wait for finish (1200 down to 0) .
Bake 1 hr at 80C (optional), keep in -20 or 4C.

Prehybridization (start at 3:00 pm)

Put membrane into Hyb-tube, dsRNA side face center of the tube.
Add Hyb-buffer 10-15 ml /100 cm²membrane in to Hyb-tube, tight the lid and put it in the chamber at 65C, put balance tube in and spin 1h. (If film shows a lot of background then increase the temperature to 68 C).

Hybridization

Add total labeling DNA (100 ul)) into 10-15 ml pre-Hyb-buffer and mix.
Spin at 65C or 68C overnight.

2. DNA 32-p labeling (start at 9:30 am)

Prepare boiling water and ice, take out 32-p from freezer (put bottle in water for thaw if need). Take out labeling kit except the Klenow.
Denature 1ul probe DNA fragment (50ng/ul) plus 30 ul ddH2O in boiling water for 2-10 min and quickly chill in ice.
Add: 2 ul dATP+dGTP+dTTP (mix 1ul each)

10 ul 5x buffer

2 ul BSA

1 ul Klenow

5 ul dCTP-32 (in hot bench, shield)

Final 50 ul

Incubate the reaction at Room T for 4-5 hours or overnight.
Purify the probe. Take a microspin column (Amersham), votex for 10 sec. break the bottom part put it in a tube, spin out the buffer in the column for 3 min. Put column into a new tube, add probe into column (in hot bench), spin for 5 min, prepare water for boiling and ice for chilling. Transfer the 50 ul probe from the spin tube to a new tube and boiling 3 min, chill on ice. Add 50 ul 0.02 M EDTA and mix. Then add this 100 ul purified probe into pre-hyb-buffer.

Day 3

1. Wash with low stringency wash buffer (WASH 1) 2x

Discard hyb-buffer into High-Level (HL) liq bottle (with shield).
Warm 100 ml /200 ml WASH 1 for 40 sec / 80 sec (not over 65C) and add it into small / big hyb-tube with shield.
Add 100 /200 ml tap water into another hyb-tube for spin balance.
Spin 15 min at 65C.
Discard the first wash buffer into HL liq bottle.
Wash again with WASH 1 at 65C 15 min, discard the secondary wash buffer into sink. (if lot of background, wash longer)

2. Wash with high stringency wash buffer (WASH 2) 1X

Wash membrane 1x with WASH 2 at 65C for 30 min.
Take out the membrane from hyb-tube, put on the paper towel, wrap with Saran Wrap.

3. Expose the sealed membrane to film

In the dark open the film box, inside has another paper bag (open side is opposite with the film box open side), take one film put it into expose cassette and close it. Put back the film paper bag back to the film box (open side need opposite).
Turn the light, put expose box in -80 to expose for 2 -3 days.
Develop the film at MBB develop room (next to the UV room) between 9:30 -5:30.
Turn off the light and take out the film put on the left side of the develop machine and move the film into the machine until hearing the peep and turn on the light.
Waite for about 3 min the film will come out in another side of the machine

Day 8

Strip the probe from membrane

- Boiling 0.1% SDS and add to membrane in a glass tray, wait until the SDS buffer cool down. Discard the SDS buffer into sink.
- Rinse the membrane with 2x SSC
- Put on paper towel; test the 32-p by G-counter. If still has 32-p, wash it with boiling 0.1% SDS 2x or 3x until no sounds.
- Wrap the membrane with Saran Wrap. Never let the membrane dry, otherwise it will not be stripped any more.
- Put in -20 for next probe hybridization.

Protocol 2-1 Sarah Chen (Feb12, 2005)

RT-PCR for dsRNA base on SSII protocol

First strand

2.5 ul dsRNA + 1ul Specific primer + 8.5ul H2O , boiling (12ul) 10 min, put in ice,

Run “soak 42”

Add: 8ul the following into the boiled 12 ul

1X 4X 6X 8X 10X

dNTP 1 ul 4 6 8 10

5x buf 4 ul 16 24 32 40

DTT 1 ul 4 6 8 10

RNA inhibitor 1 ul 4 6 8 10

SSIII 1 ul 4 6 8 10

Total 20ul run “ first strand” 42C 45’, 95C 5’, put into ice.

Second strand

1X 4X 6X 8X 10X

10x exten-buf 5 ul 20 30 40 50

MgCl₂1.5 ul 6 9 12 15

dNTP 1 ul 4 6 8 10

Taq(QIA) 0.5 ul 2 3 4 5

Taq extend 0.5 ul 2 3 4 5

Poly T2 0.5 ul 2 3 4 5

dH2O 36 ul 164 216 288 360

Total 45 ul /each

Add RT first strand 4 ul and specific primer 1ul, final 50 ul

Plus: Positive and Negative Control. (P-C and N-C)

P-C: use TOPO’s control DNA and primer

N-C: use H2O 4 ul instead of first strand.

Protocol 2-2 Sarah Chen (Feb12, 2005)

RT-PCR for dsRNA based on Yunjung’s protocol

First strand

2.5 ul dsRNA + 1ul Specific primer 1 (5 uM)+ random or specific primer 2, boiling 10 min, put in ice,

Add:

1 ul DTT

2 ul 5x buffer

0.5 ul 10mM dNTP

0.5 ul RNase inhibitor (40u/ul) (42C 2’)

0.5 ul SSII (25C10’, if using random primer)

1 ul free water

10 ul Total

Set Soak 42 file: 42C 50 min, 4C or ice (better to take out and put into ice)

Second strand

1X 4X 6X 8X 10X

10x exten-buf 5 ul 20 30 40 50

MgCl₂1.5 ul 6 9 12 15

dNTP 1 ul 4 6 8 10

Primer 1 0.5 ul 2 3 4 5

Primer 2 0.5 ul 2 3 4 5

Taq(QIA) 0.5 ul 2 3 4 5

Taq extend 0.5 ul 2 3 4 5

dH2O 30.5 ul

Total 40 ul /each

Add RT first strand 10 ul and (or specific primer, final 50 ul)

Plus: Positive and Negative Control. (P-C and N-C)

P-C: use TOPO’s control DNA and primer

N-C: use H2O 4 ul instead of first strand.

Protocol 2-3 Sarah Chen (Feb12, 2005)

RT-PCR for dsRNA modified from Yunjung’s and others’ protocols

First strand

2.5 ul dsRNA + 1ul Specific primer 1 (5 uM or 10 uM) + random (50X or 100x) or specific primer 2 (5 uM or 10 uM) , 1.25 ul f- water, boiling 10 min, put in ice,

Add:

1 ul DTT

2 ul 5x buffer

0.5 ul 10mM dNTP

0.5 ul RNase OUT (40u/ul) (42C 2’)

0.5 ul SSII (25C 10’, if using random primer)

10 ul Total

Set “Soak 42” file in old PCR machine:

(Optional: 42C 2min, pause, add SSII ),

25C 10 min if use random primer,

42C 50min,

70C 15min (stop reaction),

(Optional: add 1 ul RNase H 37C 20 min to remove RNA).

(Optional: 65C 60 min for annealing),

1. (Optional: pause, add 4ul 5x buffer (Amersham, labeling kit), 0.5 ul BSA, 0.5 ul dNTP, 0.5 ul Klenw and 4.5 ul water, 72C 5’. Put into ice).

2. (optional: add PCR buffer and others to 10 ul first strand cDNA except primers, 72 C 5 min. then add primers. Run PCR

Second strand

1X 4X 6X 8X 10X

10x exten-buf 5 ul 20 30 40 50

MgCl₂1.5 ul 6 9 12 15

dNTP 1 ul 4 6 8 10

Primer 1 0.5 ul 2 3 4 5

Primer 2 0.5 ul 2 3 4 5

Taq(QIA or taq Gold) 0.5 ul 2 3 4 5

Taq extend 0.5 ul 2 3 4 5

dH2O 30.5 ul

Total 40 ul /each

Add RT first strand 10 ul and (or specific primer, final 50 ul)

Plus: Positive and Negative Control. (P-C and N-C)

P-C: use TOPO’s control DNA and primer

N-C: use H2O 4 ul instead of first strand.

Protocol 2-4 Sarah Chen (March, 2007)

(This protocol have been used from 2006 March)

RT-PCR for total dsRNA and dsRNA fragment

First strand ( PCR tube in ice box, prepare boiling water)

2.5 ul t-dsRNA (or dsRNA fragment, hard to get bands)

1ul Primer1 (specific primer1, 5 uM or 10 uM)

1ul Primer2 (random primer 50X or 100x or specific primer 2)

1.5 ul f- water

---------

6ul Boiling 10 min, put in ice,

Add: (in another PCR tube)

1 ul DTT

2 ul 5x buffer

0.5 ul 10mM dNTP

(0.5 ul) (RNase OUT, inhibitor, optional) (40u/ul) (42C 2’)

0.5 ul SSII (invetrogen) * control tube do not add SSII, add H₂O.

-----------

4 ul

Spin 6ul boiling tube, add 6ul to 4ul, totally 10ul.

Set PCR machine as following: (file : “YP 1^st”in new PCR machine)

42C 50’> 70C 10’>95C 5’> 4C 30’ (better to put in ice directly after 95C)

Second strand

1X 4X 6X 8X 10X

10x exten-buf 5 ul 20 30 40 50

MgCl₂1.5 ul 6 9 12 15

dNTP 1 ul 4 6 8 10

Taq(invetrigen) 0.5 ul 2 3 4 5

Taq extend 0.5 ul 2 3 4 5

-------------- -------------

8.5 ul 8.5 ul /each

f-H₂O 30.5 ul 30.5 ul /each

-------------- -----------------

39 ul 39ul /each

Primer 1 0.5 ul

P rimer 2 0.5 ul

----------

Total 40 ul Add: 10 ul of RT first strand, final volume is 50 ul

New PCR (file: 2^nd YP), 94C 3’ >94C 1’ >55C 1’ >72C 2’ > &2C 15’ >4C , 45 cycles

RT-PCR Working Form Date_______________

	1	2	3	4	5
dsRNA 2ul
Primer1 1ul
Primer2 1ul
H₂O 2ul

Boiling 10 min

RT-PCR Working Form Date_______________

	1	2	3	4	5
dsRNA 2ul
Primer1 1ul
Primer2 1ul
H₂O 2ul

Boiling 10 min

RT-PCR Working Form Date_______________

	1	2	3	4	5
dsRNA 2ul
Primer1 1ul
Primer2 1ul
H₂O 2ul

Boiling 10 min

RT-PCR Working Form Date_______________

	1	2	3	4	5
dsRNA 2ul
Primer1 1ul
Primer2 1ul
H₂O 2ul

Boiling 10 min

Protocol 3 See Protocol 3 exl file Sarah Chen (Feb 12, 2005)

(from Tania)

RAPD

Reagents

1x final 10x

2.5 ul DNA (0.8ng/ul) 25

2.5 ul primer (1.5 uM) 25

1.5 ul 10X dNTP mix (2.5 mM each) 15

1.725 ul MgCL2 (25 mM) 17.25

1.5 ul 10x buffer (100 mM Tris pH 8) 15

0.08 ul Taq (5 U/ul) 0.8

5.195 ul dH2O 51.95

15 .0 ul total 15 ul/each

94 C 2 min

94 C 1 min

36C 1 min

72C 2 min

44 cycles

72C 10 min

Gel electorphoresis:

Add 3 ul of 6x loading buffer to tube. Run 18 ul in a 1.5% agarose gel containing 1 ug/ml EtBr. Use TAE running buffer to make the gel and run the gel. 5V/cm for 1.5 hours

RAPD

(from Nitin, modified by Sarah)

1x 7x 10x final

10 x PCR buffer (–MgCl2) 2.0 ul 14 20 1x

50 mM MgCl2 0.8 ul 5.6 8 2 mM

10 mM dNTP 0.2 ul 1.4 2 0.1 mM

5u/ul Taq (invitrogen) 0.2 ul 1.4 2 0.05 u/ul

12.5 uM Primer 467 1.0 ul 7.0 10 0.625 uM

Free H2O 13.8 ul 96.6 138

DNA Sample* 2 ul 18ul/each 18 ul/each 40 ng

* DNA sample from rapid fungi extraction needs to be diluted 100-1000 times, then RAPD start to work.

File RAPD sun under Yunjung user

Edit to :

94C 5’ > 94C 1’, 32C 1’, 72C 2’ >72C 8’ >4C

Check 5 ul +SBG 1 ul (1KB plus ladder +1 ul SBG) on 1 - 1.5 % agarose gel with TAE

50V, 3-4 h

Protocol 4 Feb 5, 2005 Sarah Chen

Midi preparation of plasmid DNA using Wizard plus Midipreps

Reagents

LB amp 50ug/ml liquid
14 ml tubes
White colonies patched on LB amp 50ug/ml plates
Wizard plus Midipreps kit (Cat# A7640 Promega order from Fisher PA7640)

Method

Inoculate the colonies into 6 ml LB liquid, 37C o/n
Take off the lid of the 14 ml tubes and spin at 5K 5-10 min
Discard the liquid and add 400 ul cell Resuspension Sol.
Vortex vigorously
Add 400 ul cell lysis, gently shake or vortex 2 seconds, room T 3 min
Add 400 ul Neutralization Sol., vortex 2 Seconds
Transfer all to 1.5 ml tube by pouring
Spin down 5-10 min at 12,000rpm
Put column into 3ml syringe (B-D 309571 at science store), label it.
Add 1 ml minipreps DNA purification Resin (shake it before adding) into syring
Tip out up phase from the 1.5 ml tube and add into the syringe, mix with tip or shaking
Push out and take the column out of the syringe before taking out the bar
Add 2 ml wash Sol. And squeeze out the liquid
Take the column and put in 1.5 ml tube(cut the lid of the tube)
Spin 1 min at 12,000 rpm
Warm DNA/RNA free H2O in microwave 20 second with lid open
Label new tubes and add warm H2O 50 ul in to the center of the column
Move the column into the new tubes without the lid, wait 1 min and spin 1 min
Transfer the 45-50 ul to another new tubes with lid, keep in -20

Digestion of the plasmid DNA

3-5 ul plamid DNA
2 ul 10x rec 3 buffer
1 ul EcoR1
14-12 ul dH2O
37C 1 hour
Running on 0.9% agarose gel (make with TAE), 60-90 v for 30 min

Protocol 5 Sarah Chen (Feb 12, 2005)

DsRNA extraction from fungi using CF11 column

Regent:

10 X STE buffer (0.1 M NaCl, 10mM Tris-HCl (ph8.0), 1nM EDTA (pH8.0)

(for 1 liter of 10X: 58.44 g of NaCl + 15.8 g of Tris-HCl + 3.7 g of EDTA + 1 liter of water)

10% SDS

(for 100 ml: 10 g of SDS powder dissolve in 80 ml of ddH2O, up to 100 ml)

TE buffer (10 mM Tris-HCl (pH7.4), 1mM EDTA (pH8.0))

(for 50ml : 25 ml of 200 mM Tris-HCl + 250 ul of 200 mM EDTA, and 47.5 ml of ddH2O)

3M NaOAc

(for 100 ml: dossolve 40.8 g in 100 ml and adjust to pH 7.0 with acetic acid or not)

Chloroform:isoamy alcohol (24:1)
(240 ml Chl. + 10 ml Iso. In brown bottle or white bottle)

Methods:

Harvest 14 days mycelium with Miracloth using caccum filteration
Add 1 ml of 2x STE and grind mycelium with mortar
Add 3.5 ml 2x STE wash and transfer all to 15 ml tube (red lid)
Add 500 ul of 10% SDS to 4.5 ml sample
Incubate for 30 min at room temperature
Add 5 ml of chloroform/isoamyl alcohol (24:1)
Deep it for 30 min at room temperature with mixing
Centrifuge for 15 min at 8 K
Transfer aquous phage (upper part) to new 15 ml tube
Add 100% Ethanol to make 15% final concentration of total volume :

Sample: 5 ml 6 ml 6.5ml 7 ml 8 ml 8.5 ml 10 ml

100% EtOH 880 ul 1059 1144 1235 1408 1500 1760

-20 for over night or continue
Measure 0.6 g of CF11 in 15 ml tube
Add 10 ml of 85% 1x STE 15% EtOH
Incubate it at room temperature for 10 min
Centrifuge 10 min at 8 k and discard liquid
Repeat once step 13,14 and 15
Prepare 5 ml or 10 ml syringe and pack its tip 3/4 with 2 cm long glass wool using glass pipette in fume hood (wrapping finger with paper) and keep in a rack
Mix the 15% Ethanol sample (step10) and CF11 (without liquid) and incubate on ice for 10 min
Pass the sample through the syringe (push gently if pass slow)
Wash the column with 15 ml of 85% 1x STE :15% EtOH
Transfer the syringe to a new 15 ml tube
Elute dsRNA with 4 ml of 1x STE buffer
Add 400 ul of 3M NaOAc and 9 ml of 100% EtOH
Incubate either at -20 for overnight or -80 for 1 hr
Centrifuge it at room T 8K 20 min and wash with 1 ml of 70% EtOH, spin at 8K for 10 min
Dry it at room or use water pump with clean blue tips to take away the liquid
Dissolve the pellet with 100 ul TE buffer (if do RAPD or RT-PCR use EB buffer)
transfer to new 1.5 or 2 ml micro centrifuge tube and keep it -20C

Protocol 6 Sarah Chen (Feb 12, 2005)

Rapid dsRNA and RAPD DNA extraction from Fungi C. elegans

Regent:

10 X STE buffer (0.1 M NaCl, 10mM Tris-HCl (ph8.0), 1mM EDTA (pH8.0)

(for 1 liter of 10X: 58.44 g of NaCl + 15.8 g of Tris-HCl + 3.7 g of EDTA + 1 liter of water)

10% SDS

(for 100 ml: 10 g of SDS powder dissolve in 80 ml of ddH2O, up to 100 ml)

TE buffer (10 mM Tris-HCl (pH7.4), 1mM EDTA (pH8.0))

(for 50ml : 25 ml of 200 mM Tris-HCl + 250 ul of 200 mM EDTA, and 47.5 ml of ddH2O)

3M NaOAc

(for 100 ml: dossolve 40.8 g in 100 ml and adjust to pH 7.0 with acetic acid or not)

Chloroform:isoamy alcohol (24:1)
(240 ml Chl. + 10 ml Iso. In brown bottle or white bottle)

Methods:

Collect the mycelium from plate and put into 1.5 ml tube in flow hood, keep on ice.
Ground the mycelium with sterile blue pestle for 20 seconds.
Add 450 ul of 2x STE and 50 ul 10% SDS, mix and keep in room T for 20 min
Add 500 ul of chloroform/isoamyl alcohol (24:1) in fume hood, mix.
Incubate for 20 min at room temperature
Spin down at 10 rpm for 10 min
Transfer 400 ul up phase to a new tube and add 1 ml 95% ethanol and 40 ul 3M NaoAc, mix well.
Keep in -20 C overnight or -80 C for 30 min.
Spin down at 13000 rpm for 15 min.
Wash with 70% ethanol , spin at 10 rpm for 5 min
Dry at room T for 30 min.
20 ul EB buffer (from QIAGEN Kit) or Free DNA/ RNA water. (200ng/ul)
Check dsRNA 10 ul on the 08% agarose gel.
Or diluted 10 times for RAPD ( containing DNA in the sample)

Protocol 7 Sarah Chen (Feb 12, 2005)

Rapid screen white colonies

Regent:

1 X STE buffer (0.01 M NaCl, 1mM Tris-HCl (ph8.0), 0.1mM EDTA (pH8.0)

(for 1 liter of 10X: 58.44 g of NaCl + 15.8 g of Tris-HCl + 3.7 g of EDTA + 1 liter of water)

Phenol : Chloroform : isoamy alcohol (25:24:1)
(250 ml phenol + 240 ml Chl. + 10 ml Iso. In brown bottle)

Methods:

Prepare a two comb gel. Prepare 20 of 1.5 ml tube

Add 20 ul 1X STE

Use sterile yellow tip to pick up some white colonies and blue (as control) from patched plate, label the tube

Votex for 5 sec

Add 20 ul of Phenol: chloroform: isoamyl alcohol (25:24:1) in fume hood, votex for 5sec.

Incubate for 5 min at room temperature

Spin down at 10 rpm for 15 min

Load 10 ul of up phase on the gel

70v 30 min

Take a photo to find out the plasmid with the insertion, the band big than plasmid

Pick up the insertion colonies and inoculate in 6 ml +6 ul Amp 50mg/ml. Overnight

Than do the Mid plasmid preparation

Protocol 8 Sarah Chen (Aug 29, 2005)

Adding A tail to RT- PCR products

Purpose: if RT-PCR use proofreading Taq the end have no A so it can not be clone to TOPO vector, so need to add A and then clone the fragment).

Regent:

Taq polymerase, 10x buffer, dATP 10um, 50 mM MgCL2. PCR products / fragment

Methods: (JY’s)

6.5 ul proofreading PCR fragment.
1 ul 10x buffer
0.5 ul 50 mM MgCl2
1 ul Taq polymerase (Invitrogen)
1 ul dATP (2.5um)
70C 15-30min

Protocol 9 Sarah Chen (May 15, 2006)

RNA extraction from carrot leaf

Regent:

TRIzol (Inventrogen), Liq N2, Chloroform, 2-propanal, 75% Ethanol, DEPC water or Free RNase and DNase water.

Methods:

Harvest yang leaf sample , put into 2 ml tube and put tube into Liq N2 (or wrap the leaf with Aluminum foil and put into Liq N2, for smashing the sample to powder)
Grand the sample with autoclave pestle.
Add 1 ml trizol, votex and keep in ice until 18 samples done. Then the samples stay at room temperature for 5 min.
Add 250 ul Chloroform, shake and spin at 12,000 rpm 15-20 min in 4 C. (2x optional)
Take up phase and add 500 ul 2-propanal. Room T 10-15 min.
Spin at 12,000 rpm for 10 min in 4 C. Discard up phase.
Wash with 500 ul 75% Ethanol (made with DEPC), spin 5 min at 12,000 rpm in 4 C. (wash with 95% EtOH, optional)
Dry samples at room T for 20-30 min (completely dry, otherwise hard to dissolve. ?? may be opposite)
Add 50 ul DEPC water (or adjust the volume based on pellet size)

Protocol 10 Sarah Chen (April 20, 2007)

Prepare Competent Cell

Buffer:

TFB1

30 mM Potassium acetate

10 mM CaCl2

50 mM MnCl2

100 mM RbCl

15% glycerol

Adjust pH to 5.8 with 1M Acetric Acid . Filter-sterilize (0.2Um) and store at room temperature.

TFB2

10mM MoPS or PIPES, Ph 6.5

75 mM CaCl2

10 mM RbCl

15% glycerol

Adjust pH to 6.5 with 1M KOH. Filter-sterilize (0.2um) and store at room temperature.

Procedure

This rubidium chloride protocol gives better transformation efficiencies than the calcium chloride procedures for most strains. The procedure is an adaptation of one described in reference 8.

Inoculate a single colony from an LB plate in 2.5 ml of LB medium. Incubate overnight at 37^oC with shaking (approximately 225 rpm).

on the following day, use the entire overnight culture to inoculate 250 ml of LB medium containing 20 mM MgSo₄ (this results in a1:100 dilution. Grow the cells in a 1L flask until the A600 reached 0.4-0.6 (typically 5-6 hours). A 1L FLASK IS NECESSARY FOR PROPER AERATION DURING GROWTH.

Pellet the cells by centrifugation at 4,500x g for 5 min at 4C. for a 250 ml culture, use two 250 ml centrifuge bottles in a large rotor (e.g., Sorvall GSA).

Gently resuspend the cell pellets in 0.4 volume(based on the original culture volume) of ice-cold TFB1. for a 250 ml subculture, use 100 ml of

TFB1 (50ml/bottle). Combine the resuspended cell in one bottle. For the remaining steps, keep the cells on ice and chill all pipets, tubes and flasks.

Incubate the resuspended cells on ice for 5 min at 4C

Pellet the cells by centrifugation at 4,500xg for 5 min at 4C

Gently resuspend the cells in 1/25 of the original culture volume of ice-cold TFB2. for a 250 ml subculture, use 10ml of TFB2. (Note: treat the competent cells gently as they are highly sensitive to handling and elevated temperature).

Incubate the cells on ice for 15-60 min and then aliquot 200 ul/tube for storage at -70C. Quick-freeze the tubes in a dry ice/isopropanol bath. Most cells prepared this way are stable for at least 3 months. (1 year still OK)

Protocol 11 Sarah Chen (June 27, 2008)

TOPO Cloning

SOC medium (100 ml)

2.0 g Bacto-tryptone

0.5g Bacto-yeast extract

1ml 1M NaCl

0.25 ml 1M KCl

1ml Mg2+ stock (1M MgCl2.6H2O, 1M MgSO4.7H2o), filter-sterilized

1ml 2M glucose, filter sterilized

Add Bacto-trytone, Bactr-yeast extract, NaCl and KCl to 97 ml deionized water. Stir to dissolve. Autoclave and cool to room temperature. Add 2M Mg2+ stock and 2M glucose stock, each to a final concentration of 20 mM. Filter the completemedium through a 0.2 um filter unit. The pH should be 7.0

Topo ligation reaction

3 ul PCR product or PCR fragment

1 ul Salt Sol.

1 ul (1.3ul) H2O

1 ul (0.7ul) TOPO vector

Room T 5-20 min

Add 2 ul (10 ng DNA) into 1 tube of E coli. Do not pipette, stir mix. Keep on ice for 5-30 min (note: the volume of DNA added to the competent cell must not exceed 10 ul. Optimal transformation efficiencies are observed with up to 10ng of DNA, larger quantities may cause a decrease in efficiency with some competent cell prepatations)

Put tube in 42C for 30 sec to 2 min. put in ice for 2 min.

Add SOC media 200-250 ul into tube in flow hood. (or add 3ml of LB medium

Shake at 150 rpm at 37C for 45 min to 1 hour(no more than 1 hour, satellite cell).

Add 40 ul X-gal onto LB plate )km50 or Amp100) and spread. Open the lid and dry in the flow hood.

Add 50 or 100 ul E coli culture into above plate and spread.

Put the plate in 37C overnight. (then keep in 4C overnight, some white colonies might change to blue)

Pick up the white colonies and patch on above plate ( inoculate if agent), 37C overnight.

Then do the rapid screen (optional).

Do the inoculation to LB liq Km50 or Amp100.

Extract Plasmid DNA with Wizard mid-prep kit, using 3ml syringe. Elute in 50 ul warm f-H2O

Digest P-DNA with EcorI with 4 ul DNA, 2 ul 10X rec3 buffer, 1 ul EcoRI or 0.2 ul HC-EcoRI (High consentration) , add H2O to 20 ul, 37oC 1-1.5 hours. Loading on 0.9% agarose gel. Check th e size of the insertion.

Send 10 ul to Macrogen for sequencing (account: Xiaobang)

pGEM-easy cloning:

In a PCR tube,: 2 ul RT-PCR fragment

5ul 2X buffer (votex before use)

1ul T4

0.5ul pGEM-easy vector *

1.5ul H₂O

4^oC o/n (or 16^oC for 2-4 hour , o/n)
Spin down and take 2 ul add to 50 ul competent cell (Topo cell or its made)
Ice 30 min
42^oC 1 min > ice 5 min
1 ml LB liq > 37^oC 50 min (no more than 1 hour, otherwise lots of satellite cell)
Spin down 10g 1 min, discard up and resuspend with new LB liq
Spread 10 -30 ul on X-gal plate (LB Amp100 only, Amp175 maximum, not on Km plate)
Same as TOPO clonig step 9

*pGEM-easy vector using EcoRI digestion, but pGEM vector using BamH1 and EcoRI?