View source for Rima Shah: my lab notebook

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1/30/08



== Pipetting ==



Exercise 1: 2μl blue solution + 5μl clear

Exercise 2: 20μl blue solution + 70μl clear


== Creating Solutions ==


Create 0.3M KCl solution

	In 0.3M, 0.3 mol/L

	KCl FW= 74.56g

	FW KCl * 0.3M= 22.368 g/L

For solution to fit in provided container, make 100 mL solution
	
	(22.368g/L) / 4= 2.2368g/100mL


== Plating ==


Ingredients LB/Amp plates:

	25g LB
	15g Agar
	800 mL water

Preparation:
*Dissolve 25g LB in 800mL water by swirling tube→ make final volume 1L. 
*Pour into 2L flask, add 15g Bactoagar (not agarose) and swirl until completely dissolved.
*Cover top with foil, autoclave 20-40 minutes (autoclave tape should turn black)
*Swirl solution (stirrer at 7) in flask to mix molten agar.
*Cool solution to 50 C (should be able to hold bottom 10-20 seconds)
*Add 100mg Ampicillin, swirl until dissolved.
*Lay out 40 plates, unstacked.  Pour prepared solution into plates to depth of approx. 3mm. *To remove surface bubbles, briefly flame surface with Bunsen burner.
*Leave plates out at room temp. unstacked.  Wait a few hours before stacking and 24hrs before refrigerating.
*Label plates with date and Amp.
*Store plates in plastic sleeve in refrigerator.

To turn LB plates in LB/Amp plates: Mix 200 ul 5mg/ml stock ampicillin. Add drops ampicillin in several locations around plate so that it’s uniformly distributed.  Spread solution around plate with sterile glass rod and allow it to absorb into plate. 


== Cell Culture ==


Materials:
LB-AMP plate
Sterile tubes (loose caps allow in O2)

Know culture is successful if culture becomes cloudy after 1 day

Control: LB-AMP
Culture: Plasmid PRS305 +LB-AMP + bacterial culture

Procedure:
*Label tubes: control/test, date, initials
*Fill ea. tube with LB 
*Sterilize cell loop- dip in ethanol and briefly run through fire
*Don’t’ open cell plate completely, scoop SMALL amount into tube
*Incubate: 37 C overnight—then slow shaker 12-14 hours

==Restriction Digest protocol==

DNA Digest Procedure:

*	Larger part is always insert (reporter protein etc.) and double digest is used to cut it out of plasmid
*	Smaller part remains in vector plasmid and double digest creates place for insert

XbaI/PstI DOUBLE DIGEST (used to remove insert from plasmid)
In eppendorf tube:

1)	5 ul BSA
*	BSA- Bovine Serum Albumin- stabilizes enzymes and prevents enzymes from adhering to reaction vessel

2)	5 ul Buffer H
*	Buffer H- Simulates natural environment of enzymes.  Each enzyme combination requires unique buffer with different salt concentrations.*

3)	21 ul DNA (200 ng-1ug)
*	The DNA plasmid that contains the reporter (or any protein insert)

4)	1.5ul XbaI
*	Cuts plasmid at X site (before the protein insert)

5)	1.5 ul PstI
*	Cuts plasmid at P site (after the protein insert)

TOTAL: 34 ul

SpeI/PstI DOUBLE DIGEST (used to make room on vector for insert)
In eppendorf tube:
1)	5 ul BSA

2)	5 ul Buffer B
*	Buffer B simulates environment for these specific enzymes

3)	21 ul DNA (200ng-1 ug)
*	The DNA plasmid that will serve as the vector 

4)	1.5ul SpeI
*	Cuts plasmid at S site

5)	1.5ul PstI
*	Cuts plasmid at P site

TOTAL: 34 ul


Buffers for specific restriction enzyme combinations (Manufacturer: Promega)
Eco RI/Spe I: Multicore
Eco RI/Xba I: Multicore Buffer H
Eco RI/Pst I: Buffer H
Spe I/Xba I: Multicore, Buffer E
Spe I/ Pst I: NONE (~Buffer B)
Xba I/ Pst I: Buffer H

-	Each manufacturer has different naming schemes and combinations→ all parts and buffers should be from same company

 Restriction Site Cut Sequences
(Xba I and Spe I are compatible)

Xba I: 

T CTAGA

AGAGC T

Spe I:

A CTAGT

TGATC A

Eco RI:

G AATTC

CTTAA G

Pst I:

CTGCA G

G ACGTC

Incubation of tubes- 2 hours
*Enough time to allow plasmid DNA to be cut by enzymes