Team:China Tongji/Notebook
Notebook
1. Record
- 1.1 Plasmid Part
- 1.2 Worm Part
- 1.3 Efficiency Part
- 1.4 Equipment Part
2. Timeline
1. Record
1.1 Plasmid Part
1.1.1 JUNE -- Week 1~4 -- June 6~25[Expand]
1.1.1.1 Week1 -- June 6
June 6
1, Design the PCR primers of chR2-YFP.
1.1.1.2 Week2 -- June 8~14
June 8
1, Theamplification of CHR2-YFP (use taq PCR protocol)
2, AGE ( agarose gel electrophoresis )
3, Gel extraction of chR2-YFP.
4, Transformation of GFP, YFP, mcherry in E•coli DH5α.
June 9
1, Select a single clone of each plate. (GFP, YFP, mcherry) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.
2, Transformationof vector with pmyo-3(ppd95.77)
June 10
1, Select a single clone of plate. (pmyo-3,ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.
2, Plasmid extraction ofE.coli DH5αwith GFP, YFP and mcherry in it.
June 11
1, Plasmid extraction of pmyo-3(ppd95.77)
2, Digestion of ppd95.77 with pmyo-3 in it and chR2-YFP using BamHI and EcoRI.(digestion protocol)
June 12
1, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with pmyo-3.
2, Gel extraction of pmyo-3.
3, purification of chR2-YFP witch has been digested with BamHI and EcoRI.
June 14
1, Transform of GFP, YFP, mcherryin E•coli BL21.
1.1.1.3 Week3 -- June 15~21
June 15
1, Ligation of pmyo-2 and chR2-YFP. (ligation protocol)
2, Transformation of ligation product: pmyo3-chR2-YFP (in ppd95.77).
June 17
1, Select a single clone of plate. (pmyo-3-chR2-YFP, ppd95.77) .Put the E.coli in 4ml LB buffer and train for one night at 37℃.
2, Plasmid extraction of pmyo3-chR2-YFP.
June 19
1, Digestion of new plasmid: pmyo3-chR2-YFP using HindIII and EcoRI to check if the ligation step work or not. (it worked! Our first part is done!)
June 20
1, Transformation of pmyo3-chR2-YFP in order to get more plasmid.
2, Transformation of pmec3-chR2-YFP, pmec3-dsred and pmec4-chR2-YFP which are offered by professor Li’s lab.
3, Save the E.coli strain with glycerinum.
June 21
1, Theamplification of dsred and pmyo-2(use taq PCR protocol---to test the best temperature for the PCR).
2, the amplification of dsred and pmyo-2(use pfu PCR protocol).
3, AGE ( agarose gel electrophoresis ) of pmyo-2 and dsred.
4, Gel extraction of pmyo-2 and dsred.
1.1.1.4 Week4 -- June 22~25
June 22
1, digestion of pmyo3-chR2-YFP(using HindIII and BamHI)
2, AGE ( agarose gel electrophoresis ) of digested vector---ppd95.77 with chR2-YFP.
3, Gel extraction of pmyo-3(PPD95.77).
4, Digest of pmyo2 with HindIII and BamHI. Digest of dsred with BamHI and EcoRI.
5, gene purification for pmyo2 and dsred.
June 24
1, Ligation of pmyo2 with chR2-YFP (in ppd95.77).
2, Ligation of dsred with pmyo3 and pmyo2(in ppd95.77)
June 25
1, Digest of pmyo2-chR2-YFP and pmyo2-dsred and pmyo3-dsred.(usingHindIII and EcoRI) To check if we had ligated it successfully.
2, AGE ( agarose gel electrophoresis ) of digested products. Analyze the result.
1.1.2 JULY -- Week 2~5 -- July 11~30[Expand]
1.1.2.1 Week2 -- July 11~14
July 11
1, Design the PCR primers of Blink gene.
2, Design the PCR primers of pttx-3.
3, Design the PCR primers of ptrp4.
4, Prepare for competent cells
July 14
1, Transformation of the Blink plasmid which was kind offered by Anna’s lab.
2, Transformation of pmyo2-chR2-YFP, pmyo2-dsred and pmyo3-dsred in order to get more plasmids.
3, GFP, YFP, mcherry transform OP50 and PA14. (OP50 and PA14 are the food of C.elegans)
1.1.2.2 Week3 -- July 15~21
July 15
1,Select single clones of plate. (pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred ppd95.77) . Put the E.coli in 4ml LB buffer and cultivate for one night at 37℃.
July 16
1, Plasmid extraction of pmyo-2-chR2-YFP, pmyo2-dsred and pmyo3-dsred.
July 17
1, Recovery of
pNP260( Pnmr-1::flox::ChR2::mCherry.) pCoS2(pnhr-79::Cre): pCoS13(posm-10::loxP::LacZ::STOP::loxP:: ChR2::mCherry::SL2::GFP) pSH116 (pdes-2::Cre)which are kind offered by Alexander Gottschalk’ lab.
2, Transformation of pNP260, pCoS2, pCoS13 and pSH116.
3, Design of the PCR primers of pttx-HindIII-F and pttx3-Xbal-R.
July 18
1, Select single clones of plate.(pNP260, pCoS2, pCoS13 and pSH116). Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.
2, Plasmid extraction.
3, Make genome DNA from worms.
4, Make LB plate and LB liquid.
5, Repeat the experiment: Prepare for competent cells: GFP, YFP, mcherry transform OP50 and PA14.
6, The amplification of Blink.
7, AGE ( agarose gel electrophoresis ) of blink PCR products. Analyze the result.
8, Gel extraction. (all at 300ng/ul)
July 19
1, Use the C.elegans genomic DNA as template and got pttx-3 with the help of Pttx3-HindIII-F and pttx-3-XbaI-R.
2, AGE ( agarose gel electrophoresis ) of PCR products. Analyze the result.
July 20
1, Transformation of P Blue plasmid.(used to make the mixture of microinjection liquid)
2, Select single clone on the culture plate.Put the E.coli in 4ml LB buffer and cultivate for 12h at 37℃.
3, Select a single clone of each plate and put each one in a tube that contain the ampicillin and 1ml LB culture media. (OP50 and PA14)
4, At 37℃ we culture them for 12h.
July 21
1, Add the mixture to a new 15ml tube and add in 4ml LB culture media.
2, Culture them for 3h until the OD number near 0.8.
3, Add 1mg IPTG in the liquid.
4, Culture them for 3h in order to gain the protein.
1.1.2.3 Week4 -- July 21~27
1.1.2.4 Week5 -- July 28~31
1.1.3 AUGUST -- Week 1~5 -- August 1~31[Expand]
1.1.3.1 Week1 -- August 1~7
1.1.3.2 Week2 -- August 8~13
1.1.3.3 Week3 -- August 15~21
1.1.3.4 Week4 -- August 22~28
1.1.3.5 Week5 -- August 29~31
1.1.4 SEPTEMBER -- Week 1~2 -- September 1~10[Expand]
1.1.4.1 Week1 -- September 1~7
1.1.4.2 Week2 -- September 9~10
1.2 Worm Part
1.2.1 JUNE -- June 20~29[Expand]
1.2.1.1 June 20~29
1.2.2 JULY -- Week 2~5 -- July 9~31[Expand]
1.2.2.1 Week2 -- July 9~13
1.2.2.2 Week3 -- July 15~21
1.2.2.3 Week4 -- July 23~28
1.2.2.4 Week5 -- July 29~31
1.2.3 AUGUST -- Week 1~5 -- August 1~31[Expand]
1.2.3.1 Week1 -- August 1~6
1.2.3.2 Week2 -- August 8~14
1.2.3.3 Week3 -- August 15~21
1.2.3.4 Week4 -- August 22~27
1.2.3.5 Week5 -- August 29~31
1.2.4 SEPTEMBER -- Week 1~2 -- September 1~15[Expand]
1.2.4.1 Week1 -- September 1~7
1.2.4.2 Week2 -- September 9~15
1.3 Efficiency Testpart
1.3.1 AUGUST -- Week 1~5 -- August 3~31[Expand]
1.3.1.1 Week1 -- August 3~7
1.3.1.2 Week2 -- August 9~14
1.3.1.3 Week3 -- August 15~21
1.3.1.4 Week4 -- August 22~28
1.3.1.5 Week5 -- August 30
1.3.2 SEPTEMBER -- Week 1~2 -- September 1~15[Expand]
1.3.2.1 Week1 -- September 1~7
1.3.2.2 Week2 -- September 8~16
1.4 Equipment Part
1.4.1 JULY -- July 1,28[Expand]
1.4.1.1 Week1 -- July 1
1.4.1.2 Week4 -- July 28
1.4.2 AUGUST -- Week 1~2 -- August 1~8[Expand]
1.4.2.1 Week1 -- August 1~7
1.4.2.2 Week2 -- August 8
2. Timeline
