IGEM:MIT/2005/Protocols

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.
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

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. (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.

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 75 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.

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
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

Purified Digest from Gel

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 75 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.

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.

Overnights

for amp resistant strains
5 mL LB per culture
25 uL 100X 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).

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)

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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)