Test Competent E. coli DH5a

Test our competent cells against Callahan competent cells

Competency was chemically induced by RbCl

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
	1:10	Too many to count	n/a
	1:100	201	1,005,000
2-17 (OD600=0.3)	1:1	Too many to count	n/a
	1:10	334	167,000
	1:100	17 ^[1]	85,000
4-18 (OD600=0.6)	1:1	Too many to count	n/a
	1:10	352 ^[2]	176,000
	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.

DH5-α transformants Batch 1 and 2:
(L-R)
Row 1: Callahan cells 1/1, 1/10, 1/100
Row 2: 4-18 cells 1/1, 1/10, 1/100
Row 3: 2-17 cells 1/1, 1/10, 1/100
Row 4: 4-17 and 2-15 cells positive and negative controls

Batch 3

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 10 times 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)?

Test Competent DB3.1 Cells

These cells were made on 7/11/08 with this protocol.

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

Competency was tested during a transformation from 7/14/08.

Results

The day after transformation. (+) control pUC18 yielded a lawn, the (-) control yielded no colonies. The results of the other transformations are included else where.

Team:Hawaii/Test Competent E. Coli

From 2008.igem.org

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