Team:Hawaii/Test Competent E. Coli

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Revision as of 23:10, 4 June 2008

Test Competent E. coli DH5a

Test our competent cells against Callahan competent cells

Methods

Materials

Transported 50uL of competent cells from Callahan Lab on ice
100uL batch 1 competent cells made at OD₆₀₀ ~0.6 (split into two 50 uL aliquots 4-18a,4-18b)
100uL batch 2 competent cells made at OD₆₀₀ ~0.3 (split into two 50 uL aliquots 2-17a,2-17b)

Procedure

Thaw plasmid on ice
Add 1uL plasmid pBluescript
Incubate plasmid + competent cells on ice for 30 minutes
Heat shock 60 sec at 42C
Add 250 μl SOC
Incubate at 37 C for 1 hour in 2 ml eppendorf tubes, with shaking (150 rpm)
Plate 20 uL dilutions 1/1, 1/10, 1/100 on LB+amp to test competency

Results

**Colony Count of pBluescript Transformants**
Source DH5-α	Dilution	Colony Forming Units on Plate	Number of Transformed Cells per mL
Callahan lab	1:1	Too many to count	n/a
Callahan lab	1:10	Too many to count	n/a
Callahan lab	1:100	201	1,005,000
2-17 (OD600=0.3)	1:1	Too many to count	n/a
2-17 (OD600=0.3)	1:10	334	167,000
2-17 (OD600=0.3)	1:100	17 ^[1]	85,000
4-18 (OD600=0.6)	1:1	Too many to count	n/a
4-18 (OD600=0.6)	1:10	352 ^[2]	176,000
4-18 (OD600=0.6)	1:100	40	200,000
4-17 (OD600=0.6)	1:1 (negative control)	0	0
2-15 (OD600=0.3)	1:1 (negative control)	0	0
4-17 (OD600=0.6)	1:1 (positive control ^[3])	Bacterial lawn	n/a
2-15 (OD600=0.3)	1:1 (positive control ^[3])	Bacterial lawn	n/a

Notes:
¹ Fewer than 30 colonies results in a statistically inaccurate estimation of cell density. CFU should be between 30 and 300.
² Colonies formed in one quadrant (88) were counted and multiplied by 4 to give final cell density.
³ Positive control was plated on plain LB plates, without amp.

Discussion

Next time, remember to aliquot out and transform only 50 ul of the competent cells.
Number of transformants do not appear to differ between cells grown to OD600=0.3 and OD600=0.6 prior to induction of chemical competency
Callahan lab cells appear more competent. Did he start with more competent cells (higher cell density)? Does exponential growth of E. coli seed stocks at 23C (Callahan cells, recommended protocol) result in higher competency than growth of E. coli at 37C (our cells)?

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

@@ Line 37: / Line 37: @@
 | 2-17 (OD600=0.3)||1:10|| 334 || 167,000
 |-
-| 2-17 (OD600=0.3)||1:100|| 17 <sup>1</sup> ||85,000
+| 2-17 (OD600=0.3)||1:100|| 17 <sup>[1]</sup> ||85,000
 |-
 | 4-18 (OD600=0.6)|| 1:1|| Too many to count||n/a
 |-
-| 4-18 (OD600=0.6)|| 1:10|| 352 <sup>2</sup>||176,000
+| 4-18 (OD600=0.6)|| 1:10|| 352 <sup>[2]</sup>||176,000
 |-
 | 4-18 (OD600=0.6)|| 1:100|| 40||200,000
@@ Line 49: / Line 49: @@
 | 2-15 (OD600=0.3)||1:1 (negative control)||0||0
 |-
-| 4-17 (OD600=0.6)||1:1 (positive control <sup>3</sup>)|| Bacterial lawn||n/a
+| 4-17 (OD600=0.6)||1:1 (positive control <sup>[3]</sup>)|| Bacterial lawn||n/a
 |-
-| 2-15 (OD600=0.3)||1:1 (positive control <sup>3</sup>)|| Bacterial lawn||n/a
+| 2-15 (OD600=0.3)||1:1 (positive control <sup>[3]</sup>)|| Bacterial lawn||n/a
 |}
 ''Notes:<br> <sup>1</sup> Fewer than 30 colonies results in a statistically inaccurate estimation of cell density. CFU should be between 30 and 300. <br><sup>2</sup> Colonies formed in one quadrant (88) were counted and multiplied by 4 to give final cell density.<br> <sup>3</sup> Positive control was plated on plain LB plates, without amp.
 === Discussion ===
 * Next time, remember to aliquot out and transform only 50 ul of the competent cells.