Team:Tsinghua-D/Notebook.html

2012.8.14
·Copy the eGFP gene from the standard part BBa_I714891 by the first round of PCR
·but the result is negative, maybe due the little amount of DNA template
·Prepare the transformation

2012.8.15
·Transform the BBa_I714891 to the bacterial
·Pick the single clone

2012.8.16
·Try to detect the plasmid before cultivating
·We give up this detecting method and wait for the bacterial to grow in large amount

2012.8.17
·Extract the plasmid from the growing bacterial, getting 100ng/ul,for 50ul
·Using digestion method with Pst1 and EcoR1, but he band is dark and unclear
·Using the plasmid as template to do the first round of PCR
·designing the annealing temperature gradient

2012.8.18
·Purifying the PCR product of the first round
·Repeat the PCR experiment
·Electrophoresis shows much disorder bands

2012.8.19
·Use the purified PCR product of the first round to do the second round PCR
·The designed temperature gradient proves that 61℃ is the best annealing temperature

2012.8.20
·Using the unpurified PCR product of the second round to do the third round
·Electrophoresis shows much disorder bands

2012.8.21
Purifying the PCR product of the second round by gel extraction

2012.8.22
·Conduct the amplification from 1st-round PCR product to itself to get more material ，of the 2nd and 3rd riboswitch
·Advance from 1st-round PCR product to 2nd-round PCR product by several trials of PCR system of the 2nd and 3rd riboswitch
·Try to purify the 1st-round and 2nd-round PCR product

 

2012.8.23
·Do the PCR with the purified PCR product of the first and the second round
·The designed temperature gradient proves that 59℃ is the best annealing temperature for the third round
·Double digestion of the plasmid and the PCR product with EcoR1 and Pst1
·Digestion for 37℃,2h
·Ligation of the two digestion DNA

2012.8.24
·Testing the constructed the plasmid with double digestion
·The acquired band is not corresponding to the supposed one
·We have to retry the ligation
·keep trying the PCR advancement of GFP signal part

2012.8.25
·Replicating the pSB1C3 plasmid with the protocol of iGEM
·Acquire pET-Duet plasmid as the future expression vector
·Ttart to prepare the paralleled lysosome signal part
·Get the 2nd-round PCR product of riboswitch advancing and purify the product
·The transformation results in not so good, bacterial colony grows too tiny and thick ,but we still think it will work

 
2012.8.26
·Retry the PCR for the second and the third round
·The primer may out of work
·Reorder the primer to be synthesized
·Pick the signal and positive colony of 1st riboswitch
·Run a testing gel of the colony we get
·A senior fellow apprentice challenge our PCR system setting and we start to desire and explore our system

2012.8.27
·Successfully get the 3rd round of the PCR product finally
·Purifying the product, 16.9ng/ul
·Form three groups to explore and determine the most suitable and efficient PCR system
·The lysosome testing part conduct PCR from 1st-round PCR product to 2nd-round PCR

2012.8.28
·Try to increase the amount of the PCR product
·Cascade appears in the electrophoresis
·Desire and test the PCR system considering the reagent , the instrument and the proportion of template and primer

2012.8.29
·Double digestion for the 3rd PCR product , BBa_I714891 plasmid, pEF-Duet
·Gel extraction and purifying
·analyze all the experiment already done and summarize a most suitable system and proportion and finally reach an agreement about the PCR
·prepare for the in vitro transcription from both theory and reagent

2012.8.30
·Ligation for overnight
·Do the transformation of the two ligation system
·Design the primer for the second and the their RNAT sequence
·Use the newly determined system and PCR protocol to push our riboswitch 2nd and ricoswitch 3rd step by step
·Replenish researching material ,such and plasmid provided by IGEM with GFP

2012.8.31
·Pick the single clone
·Extract the plasmid from the bacterial
·Double digestion for the two extracted plasmid
·The RNAT-eGFP-Duet is not correct
·The RNAT-eGFP-pSB1K3 is successfully constructed
·Learn how to detection fluorescence signal and choose the most dependable machine
·The RNA in vitro transcription part starts to move on and conduct the first try，however the result is random and makes no sense

2012.9.1
·Sending the plasmid for sequencing
·Pick other clone from the supposed RNAT-eGFP-Duet plate
·The lysosome part group deal with the synthesized DNA with PCR method
·The RNA in vitro transcription group adjust their method and finally get a better result
·GFP group gets fine 2nd-round PCR product

2012.9.2
·The RNA in vitro transcription group make another try of the in vitro transcription and choose another better dyeing method
·The GFP signal group keep on advance the three rounds PCR
·Ligate the GFP with PET-Duet and pSB1K3
·Send the purified plasmid for sequencing

2012.9.3
·The lysosome part group start to conduct the 1st PCR, which is parallel to the previous wok done by GFP group
·The 2nd riboswitch get reliable product
·The sequencing ending with no signal

2012.9.5
·The 3rd riboswitch also gets reliable enough products
·As for the 2nd riboswitch ,we tried to conduct digestion and ligation, but the digestion product’s purification failed
·Referring to relating material and prepare for another plasmid sequencing

2012.9.8-10
·Repeat the step of digestion, gel running, purification and ligation of riboswitch 2&3 ， but there isn’t bacterial signal colony , either.
·Take efforts to improve the purification method and change a kit
·Set different parallel experiment to verify the fluroscence

2012.9.11-9.16
·Take efforts to conduct the digestion, ligation and transformation of 2nd and 3rd riboswitch , finally grows fine bacterial colony and get out final plasmid

2012.9.17-9.22
·Incubate the transformed E-coli in 30 centigrade till it grows into stationary phrase .Then transfer them into 30. 37 and 45 centigrade and keep samples once an hour and detecting the fluorescence signals
·Operate the RNA in vitro transcription and purified the RNA products. Incubate the RNA samples in different temperatures according to our software design and then run the sequencing gel

2012.9.23-26
·Set about to conduct the new one-round PCR method of 9th and 10th riboswitch sequence, successful with 10th but failed in 9th .
· Prepare the final standard plasmid pSB1C3 and finish the sending parts submitted in pSB1C3