Notebook

Elements of the endeavor.

Construction of gRNA replaceable 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids

2014/8/30

Materials

Enzyme: BbsI, XbaI, FspI
Buffer: Buffer2.1, Cutsmart Buffer
T4 DNA ligase, T4 DNA ligase buffer
The usual things needed for transformation

Methods

Restriction digestion of pX330 (with BbsI) & 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids with XbaI and FspI

Vector	DNA	FspI	XbaI	Buffer	ddH2O	Total
0X mCherry(M)+px330 (453.7ng/ul)	6	2.5	2.5	1.5	2.5	15
2X mCherry(M)+px330 (644ng/ul)	4	2.5	2.5	1.5	4.5	15
5X M+U2(130.5ng/ul)	17	2.5	2.5	2.5	0.5	25
5X 8.28_1(300ng/ul)	7	2.5	2.5	2	6	20
5X 8.28_2(230.1ng/ul)	10	2.5	2.5	2	3	20
7X BIG(461.4ng/ul)	6	2.5	2.5	1.5	2.5	15

Insert	DNA	FspI	XbaI	Buffer	ddH2O	Total
pX330(164ng/ul)	34	5	5	5	1	50

Incubate at 37C for 4 hours.

Electrophoresis and gel extraction

Electrophoresis:

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Results of Gel purification:

Vector	0X	27.4ng/ul
Vector	2X	26.6ng/ul
Vector	5X_1	19.8ng/ul
Vector	5X_2	22ng/ul
Vector	7X	26.9ng/ul
Insert	pX330	44.2ng/ul

Ligation for small fragment of pX330 (with BbsI) and big fragment of 0X/2X/5X/7X-UAS-mCherry-pX330 plasmids

Ligation system: [unit: ul]

	Vector (needed 46.25ng)	Insert (needed 56.25ng)	H2O	T4 ligase	Buffer	Total
0X	1.7	1.3	14	1	2	20
2X	1.7	1.3	14	1	2	20
5X_1	2.3	1.3	13.4	1	2	20
5X_2	2.1	1.3	13.6	1	2	20
7X	1.7	1.3	14	1	2	20

Protocol

a. incubate at RT for 15min
b. heat-inactivation at 65C for 10min
c. Transformation
Performed as protocol…
5ul ligation reaction to 50ul competent cell

Results

Except 0X group, all the other groups have colonies grown out!

Since we still have 15ul ligation reaction remaining for each group, we just transformed bacteria with 0X ligation reaction again. [10ul ligation reaction to 100ul competent cell]

Further results

Luckily, this time there were one colony growing out on the 0X plate!

Team:SUSTC-Shenzhen/Notebook/Construction-of-gRNA-replaceable-0X-2X-5X-7X-UAS-mCherry-pX330-plasmids

From 2014.igem.org