Team:Johns Hopkins/Notebook/GROUP 3: Short two way stops

GROUP 3: Short two way stops

Summary for Short Two-Way Stops Group

 Date:October 7,2008
 Status report by: James
 Part no.:BBa_K110012;11;10;13;17
 Direct transformation into Biobrick vectors has shown interesting results via analysis with PCR
 and cutting with Nsp1. The data is somewhat hard to decipher, but I am working on it...
 Media:10-4-08 digest of 10;11;13.jpg
 Media:10-04-08 PCR verification.jpg

 Date:September 15,2008
 Status report by: James
 Part no.:BBa_K110012;11;10;13;17
 Part Descriptin: Short one-way stop (BBa_K110012 was found to be a 1-way stop) 
 Short two way stops 11;13 and sapphire Fluorescent proteins 10 and 17 Summary: PCR was performed on 
 10;11;13;and 17. Phenol Chloroform extraction; REstriction digetion was thenperformed on 
 all samples. BBa_K110017 was lost in an accident. For BBa_K110010 DPN1 was added during 
 Restriction Digest to removed the original Kurt thorn source Plasmid. Cloning into Amp
 ressistant IGEM Vectors yielded ~100 colonies per plate.
 Minipreps and RE digests will be performed on these samples.

 Restriction Digestion with EcoRI and PstI was also completed on all fluorescent Proteins 
 from MiT as well as 5 clones from each attempt at assembly (YFP+12.3, and MFA1+YFP+12.3)
 Each Fluorescent Protein had no insert present. However NEB buffer 2 was used and >5 ug of DNA was used.
 The Assemblies each showed one clone or more for correct size. Note: Lane 5's faint product
 was used to make lane 14's Plasmid DNA, thus lane 14 or MFA1+YFP+12.3 clone 5 looks like half the sex detector.
 
 09-16-08.jpg

 Date: August 25, 2008
 Status report by: James
 Part no.: BBa_K110011 -> BBa_K110013
 Part Description: Short Two way Stops
 Summary: Sequence files from parts sent for sequencing showed incorrect sequences for 3/4 biobricks sent in. However 1 clone
 of BBa_K110012 was correct. That clone was cut and ligated into iGEM vector. A PCR will be tried again for 11-->13, however direct
 ligation into iGEM vector will be attempted to evade problems with incomplete products ligating into PGEM-T.
 The Two way stop biobrick was then attached to the end of the Fluorescent Protien Venus YFP, using iGEM standard assembly.
 This Venus YFP had come from iGEM headquarters, as we as still waiting for sequence verification of our biobricks.
 We have yet to run the product on the gel, but the expected band length should be around 900bp. 
 (700bp Fluorescent Protein + 200bp Two way stop)

 Date 8/12/08
 Status report by: Raghav and James
 Part no.:BBa_K110011 -> BBa_K110013; BBa_K110010;17
 Part Description:
 Summary:Short two way stops should be sent out for sequencing this week Needed 
 so much DNA to visualize one band of short two way stop. Sapphire FP progress
 has slowed, due to extraneous bands in gel and extraneous sequence from the trace 
 files. transformations from the biobrick book were tried again with very poor
 results... 1 COLONY... POSSIBLY  THE BIOBRICK BOOK IS UPSETTING.

 Date 8/5/08
 Status report by: James
 Part no.: BBa_K110011 -> BBa_K110013; BBa_K110010;17
 Part Description: Short two way stops; and Sapphire Fluorescent Proteins
 Summary: The sequence data from last week, turned out all right
 both orientations of Sapphire FP should be correct sequences 
 Sequence Data  HOORAY!
 Last weeks miniprep and digestion yielded no DNA, so this week the minprep was repeated and the
 digestion of the prodeuct can be found here RE digest [http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-8-5.Two%20way%20stop%20digestion%208/05/08.Jasper;%20James;%20Raghav.html Two way stop digestion 8/05/08]

 Date 7/29/08
 Status report by: James
 Part no.: BBa_K110011 -> BBa_K110013; BBa_K110010;17
 Part Description: Short two way stops
 Summary: Sequencing of BBa_K110010;17 (Sapphire FP) revealed confusing, if not bad
 sequences.Sequence Data  
 PCR cloning and transformation was completed sucessfully last week on the Short two way stops
 A Miniprep was done and a  RE digest will be completed soon (possibly tonight)

 Date: 7/22/2008
 Status report by: James
 Part no.: BBa_K110011 -> BBa_K110013
 Part Description: Short two way stops
 Summary: RE digestion showed no insert the plasmid of our clones, thus the PCR was done again.
 [http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-7-23.Short%20way%20stops.James.html Short way stops]

 Date: 7/21/08
 status report by: James
 Part no.: BBa_K110011
 Part Description: Between-bud 27-W FRS2-C LtR
 Part Location (in build a genome lab): In James and Jasper's PCR product Box,
  Stainless Steel 4 degree
 PCR successful?; Yes
 Cloning of PCR product successful: Yes 
 [http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-7-22.Short%202Way%20Stop,%20Alpha%20Promoters,%20&%20Sapphire%20FP.Allison%20Suarez%20and%20Nate%20Sotuyo%20.html Short 2Way Stop, Alpha Promoters, & Sapphire FP]
 Sequencing of cloned PCR product successful: No
 Joining of validated part to adjacent part(s) status: Not done
 Problems to be solved:
 Current status of this part: Miniprep digestion yielded no product, PCR; ligation; 
 transformation; and miniprep will be repeated by this Wednesday (07/23/08)

 status report by: James
 Part no.: BBa_K110012
 Part Description: Between STE2-W and BST1-C LtR
 Part Location (in build a genome lab): In James and Jasper's PCR product Box, 
  Stainless Steel 4 degree
 PCR successful?; Yes
 Cloning of PCR product successful: Yes 
 [http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-7-22.Short%202Way%20Stop,%20Alpha%20Promoters,%20&%20Sapphire%20FP.Allison%20Suarez%20and%20Nate%20Sotuyo%20.html Short 2Way Stop, Alpha Promoters, & Sapphire FP]
 Sequencing of cloned PCR product successful: No
 Joining of validated part to adjacent part(s) status: Not done
 Problems to be solved:
 Current status of this part: Miniprep digestion yielded no product, PCR; ligation; 
 transformation; and miniprep will be repeated by this Wednesday (07/23/08)

 status report by: James
 Part no.: BBa_K110013
 Part Description: Between-SWP82-W and EMP47-C LtR
 Part Location (in build a genome lab): In James and Jasper's PCR product Box,
  Stainless Steel 4 degree
 PCR successful?; No
 Cloning of PCR product successful: No 
 Sequencing of cloned PCR product successful: No
 Joining of validated part to adjacent part(s) status: Not done
 Problems to be solved: The PCR of this part yielded a very large product
 Current status of this part: