BIO 510 2007 SECOND HALF DAY-TO-DAY LABORATORY SCHEDULE

 

Overview:

 

Wednesday, October 17th      LAB ROOM 224

Friday, October 19th               LEC ROOM 108

Monday, October 22nd           LAB ROOM 224

Wednesday, October 24th      LAB ROOM 224

Frisday, October 26th             LEC ROOM 108

Monday, October 29th           LAB ROOM 224

Wednesday, October 31st      LAB ROOM 224

Friday, November 2nd            LEC ROOM 108

Monday, November 5th         LAB ROOM 224

Wednesday, November 7th    LAB ROOM 224

Friday, November 9th             LEC ROOM 108

Monday, November 12th       LAB ROOM 224

Wednesday, Noveber 14th     LAB ROOM 224

Friday, November 16th           LEC ROOM 108

Monday, November 19th       LAB ROOM 224

Wednesday, November 21st NO MEETING

Friday, November 23rd          NO MEETING

Monday, November 26th       LAB ROOM 224

Wednesday, November 28th LAB ROOM 224

Friday, November 30th           LAB ROOM 224

Monday, December 3rd         NO MEETING

Wednesday, December 5th    NO MEETING

Friday, December 7th             LEC ROOM 108: EXAM 2 ASSIGNMENT DISTRIBUTED

Thursday, December 13th      EXAM 2 ANSWERS DUE TO ME BY EMAIL

 

 

 

Wednesday, October 17th

 

1) Get the lap top computers assigned to your group and turn it on. 

            Group 1: computers 1 and 2

            Group 2: computers 3 and 4

            Group 3: computers 5 and 6

            Group 4: computers 7 and 8

            Group 5: computers 9 and 10

            Group 6: computers 11 and 12

            Group 7: computers 13 and 14

            Group 8: computers 15 and 16

Turn on the Vector NTI program using the short cut on the desktop to start Vector NTI.

 

A) Introduction to Vector NTI.  PM will cover material provided in your Vector NTI and Homework Assignment handout.  We’ll stop at 4:30 to allow you to set up PCR reactions and will continue with the Vector NTI introduction on the following Monday.

 

B) Do step 1 of Experiment 1 (Site-directed Mutagenesis), which is to set up a PCR reaction.

 

C) Do step 1 Experiment 2 (Random Mutagenesis), which is to set up a PCR reaction.

 

 

Friday, October 19th

 

A Overview of 2nd half of BIO 510

 

B) Overview of Experiments 1 and 2; you are expected to read the handouts provided for these experiments and understand the general goals and procedures of these experiments.

 

C) Preview of next week’s lab work schedule.

 

D) Vector NTI help session.

 

 

Monday October 22nd

 

A) Do step 2 of Experiment 1 (Site-directed Mutagenesis), which is to start the DpnI digest of part of the PCR reaction from the previous lab.  Once the digest is incubating, proceed to (B).

 

B) Do step 2 of Experiment 2 (Random Mutagenesis), which is the purification of your mutant megaprimer PCR product.  Keep the purified DNA on ice and proceed to (C).

 

C) Do step 3a through 3e of Experiment 1 (Site-directed Mutagenesis), which is transformation of XL1-Blue cells with part of your DpnI-digested PCR reaction.  While the cells are incubating in the 37 degree shaker, proceed to (D).

 

D) Do step 3 of Experiment 2 (Random Mutagenesis), which is the quantitation and gel analysis of your mutant megaprimer PCR product.  First load the gel and get it running, and then give your mutant megaprimer to the TAs for quantitation.  The TAs will take pictures of your gel and give them to you for your notebook along with the results from quantitation of your mutant megaprimer. 

 

E) Do step 4 of Experiment 2 (Random Mutagenesis), which is setting up the “EZ-Clone” PCR reaction.

 

F) Do step 3f through step 3i of Experiment 1 (Site-directed Mutagenesis), which is plating the XL1-Blue cells on selective E. coli media.

 

 

Wednesday, October 24th

 

A) Do step 5 of Experiment 2 (Random Mutagenesis), which is setting up DpnI digest of some of your EZ-Clone PCR reaction.

 

B) Do step 4 of Experiment 1 (Site-directed Mutagenesis), which is miniprep DNA isolation from six of your XL1-Blue transformants (the TAs started those cultures using colonies from your plates last night).

 

C) Do step 6a through 6h of Experiment 2 (Random Mutagenesis), which is transformation of XL10-Gold cells with your DpnI-digested EZ-Clone PCR reaction.  While the cells are incubating in the 37 degree shaker, proceed to (D).

 

D) Do step 5 of Experiment 1 (Site-directed Mutagenesis), which is setting up digests of control and miniprep DNA isolated from your XL1-Blue transformants. 

 

E) Do step 6i through 6l of Experiment 2 (Random Mutagenesis), which is plating the XL10-Gold cells on selective medium.

 

F) Record the results of your XL1-blue transformation and the TA control transformations from Experiment 1 (Site-directed Mutagenesis) before you leave.

 

 

 

Friday, October 26th

 

A) Overview of Experiment 3; you are expected to read the handouts provided for this experiment and understand the general goals and procedures of this experiment.

 

B) Preview of next week’s lab work schedule.

 

C) Vector NTI help session.

 

 

Monday, October 29th

 

A) Do step 1a through 1e of Experiment 3 (Transposon Mutagenesis), which is to set up the transposition reactions.  While the reaction is incubating at 37 degrees, proceed to (B).

 

B) Do step 7 of Experiment 2 (Random Mutagenesis), which is miniprep DNA isolation from six of your XL10-Gold transformants (the TAs started those cultures using colonies from your plates last night).

 

C) Do steps 1f and 2 of Experiment 3 (Transposon Mutagenesis), which is to stop the transposition reaction and transform DH5α cells with a portion of the reaction.  While the cells are incubating in the 37 degree shaker, proceed to (D).

 

D) Do step 6 of Experiment 1 (Site-directed Mutagenesis), which is to run your digests from the previous lab on a gel.  Give your gel to the TA when it’s done and get the photo back.  Analyze these results before leaving.

 

E) Do step 8 of Experiment 2 (Random Mutagenesis), which is setting up digests of control and miniprep DNA from your XL10-Gold transformants.  The TAs will store the digests in your -20 degree box when they are done.

 

F) Do step 3 of Experiment 3 (Transposon Mutagenesis), which is plating the DH5α cells on selective medium.

 

G) Before you leave, record the results of your XL10-Gold transformation and the TA control transformations from Experiment 2 (Random Mutagenesis).

 

 

Wednesday, October 31st

 

A) Do step 9 of Experiment 2 (Random Mutagenesis), which is running a gel on the digests from the previous lab period.  While the gel is running, proceed to (B).

 

B) Do step 4 of Experiment 3 (Transposon Mutagenesis), which is DNA miniprep isolation from six of your DH5α tranformants.

 

C) Do steps 10 and 11a through 11f of Experiment 2 (Random Mutagenesis), which is transformation of A. nidulans with two of your miniprep DNAs from XL10-Gold tranformants (choose the two plasmids that are the correct size and are the brightest bands on the gel).

 

D) Do step 5 of Experiment 3 (Transposon Mutagenesis), which is setting up digests of control and miniprep DNA isolated from your DH5α tranformants.  When the digests are done, store them in your -20 degree box.

 

 

Friday, November 2nd

 

A) Overview of Experiment 4; you are expected to read the handouts provided for this experiment and understand the general goals and procedures of this experiment.

 

B) Preview of next week’s lab work schedule.

 

C) Vector NTI help

 

 

Monday November 5th

 

A) Do step 1 of Experiment 4 (Cre-lox recombination), which is setting up Cre recombination reactions.  While the reactions are incubating, proceed to (B).

 

B) Do step 6 of Experiment 3 (Transposon Mutagenesis), which is running your digests from the previous lab on a gel.  Give your gels to the TAs and get a photo back.  While your gels are running, proceed to (C).

 

C) Do step 2 of Experiment 4 (Cre-lox recombination), which is transformation of DH5α with some of the Cre-recombination reaction from the previous lab.  While the cells are incubating in the 37 degree shaker, proceed to (D).

 

D) Do step 7a of Experiment 3 (Transposon Mutagenesis), which is setting up double digests of one of your clones with a transposon hop into the pNIG6 insert.  While the digests are incubating, proceed to (E).

 

E) Do step 3 of Experiment 4 (Cre-lox recombination), which is plating your DH5α cells on selective medium. 

 

F) Do step 7b of Experiment 3 (Transposon Mutagenesis), which is running your double digests from above on gel.  Give your gels to the TA and get a photo back.  While your gels are running, proceed to (F).  If time is short, the TAs can stop your gel and have the photo ready for you by the next lab period.

 

G) Before leaving, do step 11g of Experiment 2 (Random Mutagenesis), which is patching A. nidulans transformants onto two plates. 


 

 

Wednesday November 7th

 

A) Do steps 4 and 5 of Experiment 4 (Cre-lox recombination), which is DNA minipreps and digests of your DH5α clones (the TAs started those cultures last night using colonies from your plates).  While your digests are incubating, proceed to (B).

 

B) Do step 8 of Experiment 3 (Transposon Mutagenesis), which is transformation of Aspergillus with one of your pNIG6 clones with a transposon hop in the insert.

 

C) Do step 6 of Experiment 4 (Cre-lox recombination), which is running a gel to analyze the digests above.  While the gel is running, proceed to (D).  Tell the TAs when your gel is done and they will give you a photo of the gel.

 

D) Before leaving, do step 12 of Experiment 2 (Random Mutagenesis), which is analysis of the Aspergillus plates you patched in the previous lab.

 

 

 

Friday, November 9th

 

A) Overview of Experiment 5; you are expected to read the handouts provided for this experiment and understand the general goals and procedures of this experiment.

 

B) Preview of next week’s lab work schedule.

 

C) Vector NTI help.

 

 

Monday, November 12th

 

A) Do step 7 of Experiment 4 (Cre-lox recombination), which is transformation of one of your clones from the previous lab into BL21(DE3)pLysS cells.  The clone you use should be correct based on your digests and gel electrophoresis from the last lab period.  While the cells are incubating in the 37 degree shaker, proceed to (B).

 

B) Do step 1 of Experiment 5 (Gene Knock-outs), which is setting up PCR reactions to generate 5’ flank and 3’ flank PCR products for your UBC11 deletion construct.

 

C) Do step 8 of Experiment 4 (Cre-lox recombination), which is plating the BL21(DE3)pLysS cells onto selective medium.

 

D) Before leaving, do step 9 of Experiment 3 (Transposon Mutagenesis), which is patching your Aspergillus transformants from the previous lab onto two plates.

 

 

Wednesday, November 14th

 

A) Do step 9 of Experiment 4 (Cre-lox recombination), which is sampling and inducing expression of your BL21(DE3)pLysS culture (set up by TAs), immediately on arriving to class.  After your sample is stored in your -20 box, proceed to (B).

 

B) Do step 2 of Experiment 5 (Gene Knock-outs), which is purifying, quantitating, and running a gel on your PCR reactions from the previous lab period.  While the gel is running, proceed to (C).

 

C) Do step 10 of Experiment 4 (Transposon Mutagenesis), which is the analysis of your Aspergillus plates inoculated during the previous lab period.

 

D) Do step 3 of Experiment 5 (Gene Knock-outs), which is setting up your fusion PCR reaction.  The TAs will do step 4 (analyze your PCR reaction, purify your fusion PCR product, and quantitate the product).  The fusion PCR product will be provided to you at the start of the next lab period.

 

E) Do step 10 of Experiment 4 (Cre-lox Recombination), which is sampling your your BL21(DE3)pLysS culture. 


 

 

Friday, November 16th

 

A) Preview of the lab work schedule through November 28th.

 

B) Vector NTI help.

 

 

Monday, November 19th

 

A) Do steps 11 and 12 of Experiment 4 (Cre-lox Recombination), which is preparing samples and loading SDS-PAGE gels with your protein samples form the previous lab period.  The TAs will provide you with the 7 PM sample of your culture.  While the gels are running, proceed to (B).

 

B) Do step 5 of Experiment 5 (Gene Knock-outs), which is transformation of Aspergillus using your fusion PCR product purified by the TAs.

 

C) Do step 13 of Experiment 4 (Cre-lox Recombination), which is setting up and running a western blot of your SDS-PAGE gel.  The TAs will stop the transfer and prepare the blot for you to use in your next lab period.

 

 

Monday, November 26th

 

A) Do step 14 of Experiment 4 (Cre-lox Recombination), which is probing your western blot with anti-6HIS antibody.  While the probe and the blot are incubating, proceed to (B).

 

B) Do step 6 of Experiment 5 (Gene Knock-outs), which is purifying genomic DNA from three of your Aspergillus tranformants.  While the gel is running, proceed to (C).

 

C) Do step 15 of Experiment 4 (Cre-lox Recombination), which is washing your blot and probing it with secondary antibody.  While the probe and blot are incubating, proceed to (D).

 

D) Do step 7 of Experiment 5 (Gene Knock-outs), which is setting up PCR reactions using the genomic DNA you just isolated to determine whether any of your Aspergillus transformants carry the predicted deletion of UBC11.

 

E) Do step 16 of Experiment 4 (Cre-lox Recombination), which is washing your blot and detecting the secondary antibody bound to the blot.

 

 

Wednesday, November 28th

 

A) Do step 8 of Experiment 5 (Gene Knock-outs), which is running a gel to analyze the PCR reactions you set up last lab period.  While the gel is running, proceed to (B).  When the gel is done, tell the TAs who will get you a photo of the gel.

 

B) Do step 17 of Experiment 4 (Cre-lox Recombination), which is analysis of your western blot from the previous lab. 

 

C) Do step 9 of Experiment 5 (Gene Knock-outs), which is analysis of your gel from today.

 

D) End of lab work in BIO 510 2007.


 

 

Friday, December 7th

 

Distribution and Explanation of take-home Exam 2

 

Due by email to me at 8 AM, Thursday, December 13th.