Team:Hawaii/Notebook/2008-06- 2

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Latest revision as of 02:35, 5 June 2008

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Made Competent Cells

Grace, Margaret, Norman

Seed Stock

Tested OD600 of 1 seed stock tube @ 16 hr after innoculation

OD600 = 0.53244

Separated each 1.8 mL seed stock tube to two 0.9 mL tubes

Original cryotubes = 2 mL, which is not big enough for addition of glycerol later
Next time, perhaps use other type of tube (cryotubes don't allow air for E. coli to grow)

Incubated at 37C with shaking for additional 3 hrs

Protocol suggests 23C but growth was too slow. Also, protocol is for Mach10 cells, which grows much faster.

Added 158.8 ul glycerol to 6 seed stock tubes per compentency protocol
Added 327.8 ul glycerol to 15 seed stock tubes (26.7% glycerol) per SC's recommendation

Growth curve

Took one 0.9 mL seed stock tube prior to additional incubation and added to 90 mL SOB media
Tested OD600 every hour for 90 mL SOB mixture and 250 mL SOB mixture made yesterday

Growth until OD = 0.3 - 0.6, cells then made competant
OD600 is ideal for measuring Mach10 growth; OD550 should be used for DH5-α (citation?)

**Growth Curve of DH5-α in SOB**
Time	OD for 90 mL solution	OD for 250 mL solution
11AM	OD600 = 0.007	OD600 = 0.12
12PM	OD600 = 0.01089	OD600 = 0.22
1PM	OD600 = 0.05371	OD600 = 0.48099, OD550 = 0.5303
2PM	OD600 = 0.15752, OD550 = 0.17463	OD600 = 0.57673, OD550 = 0.63531
3PM	OD600 = 0.33753, OD550 = 0.37611	n/a

Growth of DH5-α from seed stock

Added one 0.9 mL seed stock to 90 mL SOB and checked growth (OD550 & OD600) every hour

**Growth of DH5-α from seed stock**
Time	OD 600	OD 550
1 hr	0.0272	0.03005
2 hr	reading 600	reading 550
3 hr	reading 600	reading 550
4 hr	reading 600	reading 550

Competent Cells

Competent cells prepared according to protocol based on Hanahan

Competent cells were made from stocks w/ OD600 = 0.33753 and 0.57673
250 ul chilled CCMB80 was added to each 50 ul aliquot of OD600=~0.6 to get final OD600 = 1.0-1.5

100 ul competent cells were aliquoted into each tube and stored at -80C

**OD readings for competent cells, with CCMB80 added**
Tube	OD600	OD550
1	1.1936	1.2488
2	1.1182	1.1718
3	1.1289	1.1849
4	1.0059	1.0679

Note: Desired OD600 = 1.0-1.5

Discussion

Competency test tomorrow morning
Test transformation of E. coli w/ PRL1383a Thursday if cells made are competent
Okay to grow up E. coli cells (for inducing competency) from seed stock?

Quote of the Day

"You know what's ironic? If I take my shirt off, it'll just get hotter." -An anonymous friend

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

@@ Line 14: / Line 14: @@
 * Added 158.8 ul glycerol to 6 seed stock tubes per [[Team:Hawaii/Make_Competent_E._Coli | compentency protocol]]
 * Added 327.8 ul glycerol to 15 seed stock tubes (26.7% glycerol) per SC's recommendation
+<br><br>
 ====Growth curve====
 * Took one 0.9 mL seed stock tube prior to additional incubation and added to 90 mL SOB media
 * Tested OD600 every hour for 90 mL SOB mixture and 250 mL SOB mixture made yesterday
 :* Growth until OD = 0.3 - 0.6, cells then made competant
-:* OD600 is ideal for measuring Mach10 growth; OD550 should be used for DH5-alpha (citation?)
+:* OD600 is ideal for measuring Mach10 growth; OD550 should be used for DH5-&alpha; (citation?)
+<br>
 {| border="1"
-|+ '''Growth Curve of DH5-alpha in SOB'''
+|+ '''Growth Curve of DH5-&alpha; in SOB'''
 ! Time !! OD for 90 mL solution !! OD for 250 mL solution
 |-
 ! 11AM
-| OD600 = 0.007 || OD600 = 0.12
+| align="center"|OD600 = 0.007 || align="center"|OD600 = 0.12
 |-
 ! 12PM
-|OD600 = 0.01089
+|align="center"|OD600 = 0.01089
-|OD600 = 0.22
+|align="center"|OD600 = 0.22
 |-
 ! 1PM
-| OD600 = 0.05371 || OD600 = 0.48099, OD550 = 0.5303
+| align="center"|OD600 = 0.05371 || align="center"|OD600 = 0.48099, OD550 = 0.5303
 |-
 ! 2PM
-| OD600 = 0.15752, OD550 = 0.17463 || OD600 = 0.57673, OD550 = 0.63531
+| align="center"|OD600 = 0.15752, OD550 = 0.17463 || align="center"|OD600 = 0.57673, OD550 = 0.63531
 |-
 ! 3PM
-| OD600 = 0.33753, OD550 = 0.37611 || n/a
+| align="center"|OD600 = 0.33753, OD550 = 0.37611 || align="center"|n/a
 |}
+<br><br>
+==== Growth of DH5-&alpha; from seed stock====
+Added one 0.9 mL seed stock to 90 mL SOB and checked growth (OD550 & OD600) every hour
+{| border="1"
+|+ '''Growth of DH5-&alpha; from seed stock'''
+!width="100"|Time
+!width="100"|OD 600
+!width="100"|OD 550
+|-
+! 1 hr
+| 0.0272 || 0.03005
+|-
+! 2 hr
+|reading 600
+|reading 550
+|-
+! 3 hr
+| reading 600|| reading 550
+|-
+! 4 hr
+| reading 600|| reading 550
+|}
+<br><br>
 ====Competent Cells====
 * Competent cells prepared according to [[Team:Hawaii/Make_Competent_E._Coli | protocol]] based on Hanahan
+:* Competent cells were made from stocks w/ OD600 = 0.33753 and 0.57673
+:* 250 ul chilled CCMB80 was added to each 50 ul aliquot of OD600=~0.6 to get final OD600 = 1.0-1.5
 * 100 ul competent cells were aliquoted into each tube and stored at -80C
+<br>
 {| border="1"
 |+ '''OD readings for competent cells, with CCMB80 added'''
@@ Line 53: / Line 80: @@
 |-
 ! 1
-| 1.1936 || 1.2488
+| align="center"|1.1936 ||align="center"| 1.2488
 |-
 ! 2
-|1.1182
+|align="center"|1.1182
-|1.1718
+|align="center"|1.1718
 |-
 ! 3
-|1.1289
+|align="center"|1.1289
-|1.1849
+|align="center"|1.1849
 |-
 ! 4
-|1.0059
+|align="center"|1.0059
-|1.0679
+|align="center"|1.0679
 |}
 '' Note: Desired OD600 = 1.0-1.5''
 == Discussion ==
-Transform E.coli with PRL1383a tomorrow?
+* Competency test tomorrow morning
+* Test transformation of E. coli w/ PRL1383a Thursday if cells made are competent
+* Okay to grow up E. coli cells (for inducing competency) from seed stock?
+== Quote of the Day ==
+"You know what's ironic? If I take my shirt off, it'll just get hotter." -''An anonymous friend''
 {{Team:Hawaii/Footer}}