IGEM:MIT/2005/Protocols

Cloning BB Schematic Example

Phenol-chloroform extraction

Add 1:1 ratio of Phenol-chloroform --> vortex --> spin
Remove layer of Phenol-chloroform (usually at the bottom)
Transfer top layer to a new tube
Add 1:1 ratio of chloroform --> vortex --> spin
Remove chloroform layer at the bottom
Transfer the remains to a new tube
Add 1/10 amount of Sodium acetate --> vortex --> spin
Add 2.5x amount of 100% EtOH (thats ethanol for people who don't know) --> vortex --> spin
Put in -80 degrees celsius freezer for approx 20 min. till it becomes gel-like
Centrifuge at high speed (18000 rpm) for approx 20 min.
Look for a pellet
Remove supernatant
Add a decent amount of 70% EtOH to wash salt away
invert upside down, vortex lightly if needed
Centrifuge for short amount of time to see the pellet again
Remove EtOH --> air dry --> DO NOT OVERDRY, SMELL IF ETOH IS THERE OR GONE
Resuspend in dH20 (20 microlitres)

Resuspend Primers

Centrifuge vial 1 min. to allow "dry" DNA to fall to bottom.
Resuspend primers to working concentration. 
File:Conversions.xls
Find nmoles on order form for primer.
Convert nmoles to 20 picomoles/uL to find X. (This means multiply X nmoles by 50)
Pipette X uL 1X TE into vial. 
Vortex 1 min.
Insert BLUE label-cap. Label appropriately.
Store bottom shelf -20 Freezer in PRIMER box.

PCR

1 uL each primer (-20 freezer, BLUE caps)
3 uL template DNA 
45 uL PCR Super Mix (-20 freezer)
---
50 uL rxn
[[../Denature/]], [[../Anneal/]], [[../Extend/]] in thermocycler (see links for details)
Negative Control: No template DNA

Making a Gel

1%
0.5 g agarose into blue weight boat (bench C)
50 mL 1X TAE into graduated cylinder (counter with solution stocks)
Add agarose into 250 mL sterile flask (cabinet, back of room, left)
Add TAE into flask. Swirl.
Insert Kimwipes into top of flask to prevent boiling out
Microwave till boil. Continue to swirl during time until all dissolved. (Alcove 1)
Add Ethidium Bromide to flask. (0.6uL/100mL) (Cold Room, front shelf, metal bin)
Pour into gel tray of gelbox. Let sit until hardened. 
Use TAE as running buffer.

2%
1 g agarose
50 mL 1X TAE
Same procedure as above.

1% in endy lab
12 well boxes need 30-35mL gel.
EtBr added @ ~1uL/35mL.
should have 1% agarose stock most of the time.

Test PCR on gel

5 uL PCR rxn
5 uL H20 or TE
2 uL 6x sample buffer (-20 freezer)
---
12 uL total load into 12 well gel.

Clean PCR

Use Qiagen PCR cleanup kit.

1. Add 5 volumes Buffer PB to 1 volume PCR mix.
2. Apply sample to QIAquick spin column.
3. Let sit 1' to bind DNA. Spin 1'.
4. Discard flowthrough. Place back in same tube.
5. Add 750 uL Buffer PE to wash. Spin 1'.
6. Discard flow-through. Place back in same tube.
7. Spin 1' to completely remove residual ethanol.
8. Place column in clean 1.5 mL microcentrifuge tube.
9. Elute with 50 uL Buffer EB. Let sit 1', spin 1'.
Label with PURPLE, store in Cold Room.

Ethanol Precipitation

1/10 vol of 3M sodium acetate (pH5.2)
X solution of DNA

Flick tube several times to mix.

Add 2 vol or more of ice-cold 100% ethanol. 
Vortex. 
Place in -20 freezer 30 min, or -80 freezer 15 min.
Spin 5 min. in microcentrifuge.
Add 1mL 70% ethanol (room temperature).
Invert tube several times. Spin. 
Pipette off supernatant.
Dry pellet under vacuum or Speecvac (or on the bench).
Dissolve pellet in appropriate volume with EB Buffer.
Dissolve by flicking or vortex briefly.

Digest

Enzymes should be added last. Keep enzymes in freezer until needed!!
5 uL 10X NEB Buffer #_ (1, 2, 3 according to REs used) (-20 freezer, bottom shelf)
0.5 uL 100X BSA (-20 freezer, bottom shelf)
2.5 uL each restriction enzyme (-20 freezer, bottom shelf)
X uL plasmid (at least 1-10ug in 35uL is needed)
X H20
---
50 uL digestion - in PCR tube
37°C 6 hr. (2 hrs works, too).
65°C 10'
4°C forever

Label with GREEN, store in Cold Room.

Test Digest

X% 12-well gel(s)             
5 uL cut product
5 uL H20
2 uL 6X sample buffer
---
12 uL total
Negative control with uncut product!!!

Gel Purify

20 uL cut product
4 uL 6X sample buffer (-20 freezer, bottom shelf)
---
24 uL total

Gel Extract Digest

Use Qiagen Gel Extraction Kit.
1. Excise DNA fragment from agarose gel using a clean razorblade.
2. Weigh gel slice.
3. Add 3 volumes of Buffer QG to 1 volume gel.
4. Incubate at 50°C until gel slice completely dissolves.
5. Apply sample to QIAquick spin column. Spin 1'. 
6. Discard flowthrough. Place column back into same tube.
6. Add 700 uL Buffer PE to wash. Spin 1'.
7. Discard flowthrough. Place back into same tube.
8. Centrifuge for addition 1' to remove all ethanol residue.
9. Place column in clean 1.5 mL microcentrifuge tube.
10. Add 50 uL Buffer EB, let sit 1', spin 1' to elute.
11. Label with BLUE label. Store in Cold Room (4°C).

Nanodrop Product

1. 1 uL blank (usu. EB)
2. Hit REBLANK
3. Record figure
4. Clean with H20 using Kimwipe
5. 1 uL product
6. Hit MEASURE
7. Record figure
8. Clean with H20 using Kimwipe
9. Repeat #5 and on if necessary.

Location: Endy Lab or Knight Lab
Obtain concentration (ng/uL) from Nanodrop.
Use Conversions.xls to find fm from concentration and size (bp) of DNA.
Need 20 fm recipient, 60 fm insert for ligation.

Ligation

For good results, use a 1:3 mole ratio of recipient:donor to force ligation rxn.
20 fm recipient
60 fm insert
2 uL 10X ligase buffer (-20 freezer)
0.5 uL T4 DNA ligase (-20 freezer)
X uL H20
---
20 uL total ligation
Negative controls: 1 without insert, 1 without DNA, 1 with only insert
16°C 15'
4°C forever

Label YELLOW, store in Cold Room.

note: go to Endy Digest Protocol for help figuring out gram amounts of DNA to add

Pouring Plates

Amp Plates
10mL Amp
1000mL LB
Kan Plates
1mL Kan
1000mL LB

Transformation (CaCl2 competent cells)

Protocol per tube
50 uL competent cells
3 uL ligation reaction

GENTLY mix ligation/cells with pipette tip.
Incubate on ice 10'.
Heat shock in 42°C 2'.
Immediately place on ice.
Add 1 mL LB to each tube, transfer contents to culture tube. 

Place tubes in Warm Room rollerdrum 37°C 15'.

Use sterile technique.
Plate 100 uL transformation onto LB-DrugMarker plate. 
Incubate upsidedown overnight at 37°C.
Negative Controls from ligation! 1 vector only, 1 insert only, 1 without DNA.

Another Method
        1. Thaw cells on ice.
        2. Add 2 uL rxn
        3. Let incubate on ice 30'
        4. Heat shock 42 for 30 sec.
        5. Add 250 uL SOC or LB
        6. Grow at 37 rollerdrum 1 hr.
        7. Spin 1'
        8. Pull off SOC/LB
        9. Resuspend in 100 uL SOC/LB
        10. Plate.

Even Better Method
        1. 20 uL ligation
        2. 1000 uL 1-butanol
        3. Mix
        4. -80 for 15'
        5. Spin at 13K rpm, 30'
        6. Pull off s/n
        7. Dry pellet in SpeedVac
        8. Resuspend in 10 uL H20
        9. Transform all 10 uL in 50 uL Omnimax E. coli cells.

Overnights

for amp resistant strains
5 mL LB per culture
25 uL 200X Amp per culture

for kan resistant strains
5 mL LB per culture 
5 uL 1000X Kan per culture

...continued
X culture tubes (big ones with blue cap)
Use sterile technique!!!
Use a sterile toothpick to dab the single colony desired.
Stir into culture tube of LB+DrugMarker. 
Incubate in Warm Room on rollerdrums for no more than 16 hours.

Glycerol Stocks

900 uL overnight
300 uL 40% glycerol
freezer tube, labeled legibly
-80 Freezer.

MiniPrep Plasmid

Qiagen Kit
1. Pellet cells in 1.5mL microcentrifuge tube 13K rpm, 1'.
2. Resuspend pelleted cells in 250 μl Buffer P1. No visible clumps!
3. Add 250 μl Buffer P2, invert 4-6X to mix. Do not vortex!
4. Add 350 μl Buffer N3, shake 4-6X to mix. Should be cloudy
5. Centrifuge for 10' @ 13K rpm. compact white pellet should form
6. Pipette supernatant to the QIAprep spin column. Dispose tube w/ white gunk. Spin 1'.
7. Add 750 μl Buffer PE, spin 1'.
8. Discard flowthrough, spin dry 1'.
9. Place in clean 1.5mL microcentrifuge tube.
10. Elute with 50 μl EB, spin 1'.
11. Nanodrop for concentration (ng/μl)
12. Label with RED label. Store in Cold Room (4°C).

Lambda Red

1.Grow 5mL of overnight culture at 30 degree
2.Turn on water bath to 32 degree. Turn on another water bath for 42 degree.
3.In a baffled flask, dilute 2 mL of cluture into 100 mL LB. Grow at 32 degree to an OD of 0.4-0.6
4. Make an ice water slurry.
5.In a 50 mL baffled flask, put 10 mL of this culture. Make a 10mL control.
  Put in a 42 degree shaker at 200 rev/min for 15 mins.
6.Swirl the flasks in the ice-water slurry for 10 mins. 
7.Centrifuge for 8 mins at 5,500g at 4 degree.
8.Resuspend pellet in 1 mL ice-cold sterile water. Transfer to 1.5mL tubes. Spin 20s at 4 degree in microfuge
9.Wash pellet twice with cold, sterile water.
10.Suspend in 100microL ice-cold sterile water. Store on ice.
11.Aliquot into 50microL tubes. Add desired amount of plasmid/chromosomal DNA and insert/linear DNA, for example:
   a. 0.1 ng plasmid/chromosomal DNA
   b. 300 ng insert/linear DNA
12.Pipette cells into a precooled cuvette, and electroporate.
13.Immediately after electroporation, add 1mL of SOC media.
14.Incubate at at 30 degree after electroporation.

QuikChange

*Only use QuikChange I or QuikChange II
*QuikChange II Protocol (use Ultra poly)
*QuikChange I Protocol (use Turbo poly)
*Things you'll need (can get it from TK)

Kit includes:
1. PfuUltra High-Fidelity DNA poly (25 U for 10 rxns)
2. 10X rxn buffer
3. Dpn I RE (100 U)
4. control primer 1 (750 ng)
5. control primer 2 (750 ng)
6. pWhitescript (50 ng)
7. dNTP mix (10 microlitters)  use straight from TK’s LAB
8. XL1-Blue cells (3 x 200 microlitters) 
9. pUC18 (10 microlitters)

Note: XL1-Blue needs to be stored in -80; the rest are stored in -20.
XL1-Blue and dNTP need to be kept in freezer prior to usage because
Transfering tubes from one freezer to another decreases efficiency.
Other things:'

1. 14ml Falcon polypropylene tybes
2. X-gal in DMF
3. IPTG in dwater
4. LB Amp X-gal IPTG plates
5. FecA in BB plasmid
6. FecR in BB plasmid
7. mutagenesis primers for FecA
8. utagenesis primers for FecR
9. NZY+ broth (can be replaced by LB or superbroth)

Sequencing

-In order to be sure we have what we think we have, we need to sequence our constructs.
-This is done by the Biopolymers Lab

1.[[../Fill out/]] the sequencing request form
2.For each construct that is to be sequenced, you will need two tubes: 
    One with forward primer, one with reverse
  For longer constructs (>1.5 kb) you need internal primers. 
    You can get ~800 bp of sequence info from a single primer
  Make up one reaction mixture per primer:
  100-200 ng of DNA (prepped ds plasmid DNA)
  3.2 pmoles of sequencing primer (1 ul of 20uM standard stock primer)
  ------
  final volume of 12 ul, in sterile DI water. Use tiny PCR tubes. 
  ------
  Label tubes with the number on the order form that corresponds to the sequence ID.
3.Place all tubes in a large 50 ml falcon tube; label the side of the large falcon tube with your name, 
  the date, and Endy Lab.
-Deliver tube and 1 of the 3 copies of the form to the Biopolymers Lab, E17-415. 
-Place tube in brown fridge near front of room, and request form in the receptacle on the fridge. 
-Keep one copy of the form for your records, and be sure to place the third copy in the 
 "Completed" file in the Endy lab. 

notes: 
 1. you can get the concentration of your miniprep from nanodropping and use this to get the correct amt of DNA
 2. be sure to follow the instructions in the miniprep protocol that are required for direct sequencing.
 

M9 Media

Materials

• 2X M9 (from the kitchen: this contains sodium phosphate, potassium phosphate, sodium chloride, and ammonium chloride)
• 0.1 M MgSO4 (from kitchen)
• 18% glucose (TK's lab)
• L-arginine powder
• 1M CaCl2 solution (TK's lab)
• Sterile H2O

To make 400 ml of m9, add:

• 200 ml of Fx m9
• 4 ml of 0.1 M MgSo4
• 4.44 ml of 18% glucose
• 16 mg of L-arginine
• 40 ul 1M CaCl2 solution
• 135.56 ml sterlie H2O

---

Notes from Lab Bootcamp

1. [[../Monday, June 6th/]] (Pipetman use, Working with bacteria, PCR)
2. [[../Tuesday, June 7th/]] (Miniprepping DNA, Agarose gels, RE digests)
3. [[../Wednesday, June 8th/]] (Excising DNA from gels, purifying DNA from gels)
4. [[../Thursday, June 9th/]] (Ligations, Transformations)