《PEG介导小立碗藓转化过程》由会员分享，可在线阅读，更多相关《PEG介导小立碗藓转化过程（5页珍藏版）》请在金锄头文库上搜索。
1、PEG-mediated DNA transformation into protoplasts of Physcomitrella patensStable transformation using protoplasts and PEGForeign DNA is introduced into protoplasts prepared from propagated protonemata.the isolation efficiency of protoplasts depends on the age (days from subculture) of the protonemata
2、. We obtained the best results using protonemata grown for 4 to 6 days. When older protonemata were used it was difficult to digest the cell walls using the enzyme driselase. For optimum results ,vigorous, light green protonemata should be used. Transformation scheduleWe routinely transformed proton
3、emata according to the schedule below. 1. (day 1) Subculture the propagated protonemata at 25 for approximately 4-6 days. 2. (day 5 at the earliest ) DNA transfer takes 2 days. After the transfer of DNA, incubate for 5 days. 3. (day 12) Transfer cellophane with top agar containing regenerating proto
4、plasts onto a selection plate supplemented with adequate antibiotics. Cultivate for 5 days on the selection medium. 4. (day 17 ) Transfer cellophane with moss colonies onto a non-selection plate (no antibiotics). Cultivate for 5-7 days on the non-selection medium. 5. (day 24) Transfer cellophane wit
5、h moss colonies onto a selection plate supplemented with adequate antibiotics. Cultivate for 5-7 days on the selection medium. 6. (day 24) Transplant a selection of the peripheral section of the colonies (100-200 lines)onto a non-selection plate. Cultivate for 7-10 days on the non-selection medium.
6、7. (day 41 ) Transplant a selection of the peripheral section of the colonies onto a selection plate. Cultivate for 7-10 days on the selection medium. 8. (day 51) Transplant colonies surviving on the selection medium as stable transformants onto non-selection medium.1. Propagation of protonemata(1)G
7、rowth mediumStock mediumStock B(100)： MgSO4.7H2O 2.5g Fill up to 100ml with H20 （autoclave）Stock C(100)： KH2PO4 2.5g Fill up to 100ml with H20 （autoclave）Stock D(100)： KNO3 10.1g FeSO4.7H2O 125mg Fill up to 100ml with H20 （autoclave）TES (1000)： CaSO4.5H2O 5.5mg H2BO3 61.4mgCOCL2.6H2O 5.5mg NaMoO4.2H
8、2O 2.5mgKI 2.8mg ZnSO4.7H2O 5.5mgMnCL2.4H2O 38.9mg Fill up to 100ml with H20 （autoclave）Ammounim tartrate (100)：Ammounim tartrate 9.21g Fill up to 100ml with H20 （autoclave）CaCL2(100)：CaCL2.2H20 1.47g Fill up to 100ml with H20 （autoclave）Medium used for protonemata cultureBCDAT medium 1000mlStock B
9、10ml Stock C 10ml Stock D 10ml TES 1ml CaCL2 10ml Ammounim tartrate 10ml Agar 8g Fill up to 1000 ml with H20 （autoclave）After autoclave, pour into 50 dishes of 9 cm and solidify for 30min. on a clean bench. Store at room temperature.(2)sub-cultureThe 4-6 days culture on a 9 cm dish is harvested with
10、 dental forceps, suspended in 8- 10ml of sterile water per cultured dish and blended using a nomogenizer. 2ml of suspension is inoculated in a new dish overla with cellophane.after 4-6 days of incubation at 25 under continuous white light(40umol/m2/s), growing protonemal tissue, consisting mainly of
11、 chloronemata, is obtained on the dish. The dish may be sealed with medical suegical tape to prevent contamination.MaterialSolid medium (BCDAT)-9cm-peri dish Sterile water Cellophane (autoclave)-Preparation of cellophane is as follows: 1) Place cellophane in a glass Petri dish and add 5mM EDTA solut
12、ion (pH8.0). Aotuclave. 2) Wash with milliQ water several times 3) Add milliQwater to the dish. Autoclave. Pipette man p-1000 Dental forceps Homogenizer Ultra turrax T-8(IKA) Ultra turrax T-8 generator shaft S8N-8G(autoclave) Glass tube (30-40ml)(autoclave)Procedure1. Overlay the soild medium with c
13、ellophane. 2. Add 8-10ml of sterile water (per cultured dish) in a glass tube. 3. Recover propagated protonemata from one cultured dish(4-6days old) withdental forceps and add to the glass tube. 4. Blend for about 10sec. with Ultra turrax at max speed. Used generator shaft must be washed as soon as
14、possible to prevent the adhesion of moss cells inside. 5. Inoculate 2ml of suspension into a new 9cm-petri dish.6. Incubate at 25 under continuous white light.2.1 Transformation(on the First day)Materials500 mi beaker (for discarding waste solution) 10ml disposable pipet(3) (aterile) 35ml centrifuge
15、 glass tube(2) (autoclave) Funnel with a 80um nylon mesh(autoclave) Forcers(1) Cellophane (cut into circles)(clave) Yellow tips for P200 and blue tips for P1000(autoclave) 0.22um syringe-driven filter unit and 10ml syringe(2) 0.45 um syringe-driven filter unit and 50ml syringe(1) Neubauer hemacytometer(Becton Dickinson no.424011) Wa
