tDNA 克隆:λZAP 载体编码技术说明书

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Helper Phage

The following is based on the λZAP cDNA synthesis manual from Stratagene. See also the Stockinger Lab

protocol “M13K07 Helper Phage Production.”

Background:

Stratagene provides two different helper phages with their λZAP-cDNA synthesis kit:

(1) ExAssist interference-resistant helper phage

(2) VCSM13

ExAssist is used to excise, in vivo, the pBluescript phagemid from the λ Uni-ZAP XR vector. To do this

efficiently and without contaminating the excised phagemid with the ExAssist helper phage, this

methodology utilizes the Stratagene E. coli strain SOLR. This is because ExAssist helper phage contains an

amber mutation (UAG) that requires an amber suppressor tRNA in order to grow. When plated on the

SOLR nonsuppressing strain (Su-) strain, only the excised phagemid is permitted to grow. However to

propagate (amplify) the ExAssist helper phage requires using the E. coli strain XL1-Blue MRF′ because

this strain harbors the supE44 mutation, which provides a glutamine suppressor tRNA. The VCSM13

helper phage is used strictly to produce single stranded DNA from the already excised phagemid. (This is

also what M13K07 is used for.)

Storing the Helper Phage:

Stratagene supplies both helper phages in 7% DMSO and recommends storing them at -80

o

C. As long as

there is a –80

o

C stock in the lab, the amplified lab prep can be stored at +4

o

C.

Titering the Helper Phage:

Streak out E. coli strain XL1-Blue MRF′ onto an LB

Tet15

plate.

Pick a single colony of XL1-Blue MRF′, inoculate into LB and grow to OD

600

= 1.0. (No need to include

the antibiotics in the overnight cultures.)

Meanwhile prepare a serial dilution of the phage in TE buffer. Expected number of phage should be about

10

10

pfu/ml; therefore dilute phage to create dilutions 10

-4

to 10

-7

.

Dilutions

10

-2

10

-4

10

-5

10

-6

10

-7

Phage 10µl (stock) 10µl (10

-2

) 100µl (10

-4

) 100µl (10

-5

) 100µl (10

-6

)

TE 990µl 990 900µl 900µl 900µl

Note:

When preparing serial dilutions of bacteriophage, it is usually much more accurate to dilute 10µl into 100

to create a 10

-1

dilution or 10µl into 1000µl to create a 10

-2

dilution rather than to dilute 1 into 10µl or 1µl

into 100µl because one tends to obtain grossly exaggerated titers resulting from small pipetting errors. This

is particularly critical for the first dilution.

Add 200µl of XL1-Blue MRF′ cells at OD

600

= 1.0 to individual sterile culture tubes (the number of phage

dilutions you plan to plate) in a test tube rack.

Add 100µl of each serial dilution of helper phage to the culture tubes containing the XL1-Blue MRF′ cells.

Place the test tube rack into a 37

o

C water bath for 15 minutes to allow the helper phage to attach to the

cells.

Meanwhile melt NZY Top Agarose in the microwave and allow it to cool to ~48/50

o

C. You need to pay

close attention to the bottle containing the top agarose because it can quickly boil over; alternatively use a

low power setting on the microwave.

At the conclusion of the 15-minute incubation, add ~3 ml of the NZY top agarose to the test tube

containing the helper phage & . Remove the tube from the rack (which is still sitting in the water

bath) and give it a quick flick of your wrist mixing the contents and immediately pour it onto an NZY plate.

(I usually do three plates at one by pipetting 10ml of the NZY top agarose from the bottle, dispensing ~3.3

to one tube, ~3.3 to second tube and then another ~3.3 to a third tube. I then pour the plates starting with

the first tube I put the NZY into.)

Allow the top agarose to cool (~5 minutes).

Invert the plate and incubate overnight at 37

o

C.

Count the number of plaques.

Determine the titer; i.e., pfu/ml using the formula:

(Number of plaques (pfu) X Dilution Factor) X 1000ul/ml

Volume plated (µl)

Where the volume plated (in µl) refers to the volume of the helper phage solution added to the cells.

Amplifying the Helper Phage:

Streak out E. coli strain XL1-Blue MRF′ onto an LB

Tet15

plate.

Pick a single colony of XL1-Blue MRF′ and inoculate into 10ml 2X YT and grow until OD

600

= 0.3

(~2.5 x 10

8

cells/ml). (No need to include the antibiotics in the overnight cultures.)

Add helper phage at a multiplicity of infection (MOI) of 20:1 (phage-to-cells ratio). Do this for both

ExAssist and VCSM13.

Note: (If amplifying VCSM13 helper phage, add kanamycin to a final concentration of 25µg/ml to the

medium 30 minutes after the helper phage and cells have been allowed to grow together.)

Grow the culture at 37

o

C with vigorous aeration (~300RPM) for 8 hours.

Heat the culture to 65

o

C for 15 minutes.

Spin down the cell debris and transfer the supernatant to a fresh tube.

Titer the helper phage produced.

ExAssist should be 7.5 x 10

10

to 1.0 x 10

12

pfu/ml and

VCSM13 should be 1.0 x 10

11

to 1.0 x 10

12

pfu/ml

Store at +4

o

C (Add DMSO to 7% for storage at –80

o

C).

Stockinger Lab

Notes: Several steps greatly aid the ease and success of pouring good phage onto plates:

(1) Position the 37

o

C and 48

o

C water baths right next to each other with enough room in front of them for

you to work. Wipe this area down with 70% EtOH prior to plating.

(2) Use plates that have been prewarmed to 37

o

C (for one hour or more).

(3) Use sterile glass pipettes that have been prewarmed to ~50

o

C.

(4) Have a pipette holder in the work area.

Stockinger Lab

Media:

LB

Tet15

plates

100ml

Bacto Tryptone 1.0g

Yeast Extract 0.5g

NaCl 1.0g

dH

2

O 100ml

Adjust pH to 7.5 with NaOH

Agar 1.5g

Autoclave

Cool to 48/50

o

C

Tetracycline (12.5mg/ml) 120µl

Tetracycline (12.5mg/ml)

Tetracycline 0.125g

ddH

2

O 5.0ml

EtOH 5.0ml

Filter sterilize

Store at –20

o

C

(Tetracycline is light sensitive so keep plates in the dark)

NZY

1000ml

NZ Amine 10.0g

Yeast Extract 5.0g

NaCl 5.0g

2.0g MgSO

4

·7H

2

O

dH

2

O 1000ml

Adjust pH to 7.5 with NaOH (~2 pellets)

Autoclave

NZY Agar Plates

1000ml

NZ Amine 10.0g

Yeast Extract 5.0g

NaCl 5.0g

2.0g MgSO

4

·7H

2

O

dH

2

O 1000ml

Adjust pH to 7.5 with NaOH (~2 pellets)

Agar 15.0 g

Autoclave

Cool and pour plates

NZY Top Agarose

NZY liquid medium 250ml

Agarose 1.75g

Autoclave

Stockinger Lab

2X YT

1000ml

Bacto Tryptone 16.0g

Yeast Extract 10.0g

NaCl 10.0g

1000ml dH

2

O

Adjust pH to 7.5 with NaOH (~3-4 pellets)

Autoclave

Stockinger Lab

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