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 [[Media:JHU_Saph_Seq_1.zip|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 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.[[Media:JHU_Saph_Seq_1.zip|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. 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 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 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: